BIOCHEMISTRY DISCUSSION
Leahh
13SignalTransductionPathways.pptxBiochemistry: A Short Course Fourth Edition CHAPTER 13 Signal-Transduction Pathways
Tymoczko • Berg • Gatto • Stryer
© 2019 Macmillan Learning
Created by Brett Barbaro
Ok - so now we’ve covered all of the fundamental molecules that we are going to be talking about, and now it is time to start talking about how these molecules interact with each other to make cellular processes occur. And the first thing that we are going to talk about are these signal transduction pathways. Signal transduction pathways are quite large and complex. We’re not going to go into all of the details involved with the signal transduction pathways, but I am going to show you an overview of some of the different types of signal transduction pathways. And I am hopefully going to give you enough information so that when you see a signal transduction pathway written down in a book, schematically in a diagram or something, you’ll be able to interpret it and understand what is going on. {THIS IS WHAT YOU NEED TO BE ABLE TO DO ON THE TEST}
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Chapter 13: Outline
13.1 Signal Transduction Depends on Molecular Circuits
13.2 Receptor Proteins Transmit Information into the Cell
13.3 Some Receptors Dimerize in Response to Ligand Binding and Recruit Tyrosine Kinases
13.4 Metabolism in Context: Insulin Signaling Regulates Metabolism
13.5 Calcium Ion Is a Ubiquitous Cytoplasmic Messenger
13.6 Defects in Signaling Pathways Can Lead to Diseases
Created by Brett Barbaro
All right, so there are {5} sections that we are going to be going over. We will be talking about molecular circuits in the first section, and how receptor proteins that are in the membrane can transmit information into the cell across the membrane, and then how some receptors dimerize and recruit these “tyrosine kinases”, and then we will be giving a few examples of actual processes such as insulin signaling and calcium ion signaling. And we are going to skip the last section (13.6) on defects in signaling pathways leading to diseases. I am sure you can imagine there is a number of different steps in these signaling pathways, and disrupting any of those steps can cause them to malfunction and lead to physical problems.
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13.1 Signal Transduction Depends on Molecular Circuits
Learning objective 4: Identify the basic components of all signal-transduction pathways.
Signal-transduction cascades have many components in common:
Release of a primary messenger as a response to a physiological circumstance.
Reception of the primary messenger by a receptor, usually an integral membrane protein.
Relay of the detection of the primary messenger to the cell interior by the generation of an intracellular second messenger.
Activation of effector molecules by the second messenger that result in a physiological response.
Termination of the signal cascade.
Created by Brett Barbaro
Your book breaks down the signal transduction cascades into these 5 different sections, which are pretty reasonable. There’s first the release of a primary message, and that’s from wherever is trying to send the message. If your adrenal glands are sending out adrenaline, then the adrenaline is the message. And then that message will interact with a receptor somewhere in the body, probably on the surface of a cell. Then a signal is then carried down through the receptor, which is then transmitted to the inside of the cell and amplified. Amplification, although not necessarily a part of signal transduction cascades, is nevertheless an extremely common part of signal transduction. And then, of course, the activation of the effector molecules, the actual changes that occur inside the cell. And then, of course, it all has to stop when the signal ends.
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AMPLIFICATION
SIGNAL
RECEPTION
ACTIVATION
TERMINATION
An Example of a Signal Transduction Pathway
AMPLIFICATION
<EDITED by BB>
Created by Brett Barbaro
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Figure 13.4 <EDITED> The activation of protein kinase A by a G-protein pathway. Hormone binding to a 7TM receptor initiates a signal-transduction pathway that acts through a G protein and cAMP to activate protein kinase A.
Here is an example of a signal transduction pathway, sort of a generalized signal transduction pathway - well, I mean, this one’s specific for epinephrine {but it shows the general features of signal transduction}. But this one was showing the five stages that we talked about in the last slide. Starting up in the upper left hand corner, with the epinephrine molecule, the signal coming in and binding the beta-adrenergic receptor, which is reception. And you can see the little yellow oval in there, that’s the epinephrine. And that causes a conformational change in the receptor, which then affects the conformation of the alpha protein that’s bound to it. Now you see beneath the receptor there, there’s the purple alpha, blue beta and yellow gamma subunits of the g protein trimer. {And this diagram, I think, is misleading because it has the {alpha subunit} sticking out, way out into the cytoplasm from the receptor. That purple alpha subunit is actually membrane-bound, so is the gamma subunit, so they should be actually adjacent to the membrane. – THIS HAS BEEN FIXED IN THE DIAGRAM – PLEASE REFER TO THIS DIAGRAM INSTEAD OF THE ONE IN YOUR BOOK.} Once the conformation of the receptor is changed, it changes the conformation of this alpha subunit of this trimer and allows it to release the GDP that’s bound to it, and in its place it can bind to GTP. And when it binds a GTP, then that changes its conformation again, allowing it to release from the receptor and the beta and gamma subunits, and diffuse laterally out into the membrane. Now one receptor that’s activated can actually activate several of these alpha subunits, so that’s why this is a step of amplification. You have one molecule of epinephrine going in, but it can actually activate hundreds of these {GDP}-bound alpha subunits of the G proteins. So that’s one layer of amplification.
And so they start diffusing outward - and when they hit an adenylate cyclase enzyme, they activate that enzyme, and that enzyme starts cyclizing adenylate (ATP). It turns ATP into cyclic AMP, so you can see the big black line leading down, showing that it turns ATP into cyclic AMP. And that’s only when the activated alpha subunit is bound to it. So this is another amplification step, because it doesn’t just turn one ATP into cyclic AMP, it turns hundreds, if not thousands - I don’t know what the number is, but it just basically keeps on doing it as long as the alpha subunit with the GTP attached is bound. So that’s another amplification step.
And then the cyclic AMP spreads out through the cytoplasm. So now, this is no longer membrane bound - this is something that is able diffuse throughout the cytoplasm, where it can interact with proteins such as protein kinase A. Protein kinase A is an enzyme that performs a lot of specific functions in the cell. It’s a kinase so you can guess it attaches phosphate groups to its targets, whatever they are, and causes changes to happen - and that’s the activation stage. So the cyclic AMP is basically turning on the kinase and causing it to do its job -phosphorylate its targets.
And the last stage is termination. We can go back up to the adenylate cyclase with the alpha subunit of the GTP binding protein there. And it has got the GTP bound to it - but it is a GTPase. It actually breaks down GTP into GDP, and it just takes a little while for that to happen. So until that happens, it’s activating the adenylate cyclase; but after it hydrolyzes that GTP, then it’s no longer activating adenylate cyclase - it’s no longer bound to adenylate cyclase and it can float off. It’s still membrane bound, so it diffuses laterally in the membrane, until it encounters the beta and gamma subunits that are floating around in there, and then they can all re-attach to {a} 7 transmembrane protein receptor. And if there’s still epinephrine or adrenaline (they are the same thing) still bound to the receptor, then they can start the whole thing all over again.
Section 13.2 Receptor Proteins Transmit Information into the Cell
Learning objective 5: Differentiate the various types of membrane receptors.
There are three major classes of membrane receptors:
Seven-transmembrane-helix receptors associated with heterotrimeric G-proteins.
Dimeric membrane receptors that recruit protein kinases.
Dimeric protein receptors that ARE protein kinases.
Created by Brett Barbaro
All right, so we’ve broken up the three major classes of membrane receptors into these 7 transmembrane receptors associated with heterotrimeric G-proteins. Those are the G-protein-coupled receptors, and that’s one of the biggest classes. And then, there is the dimeric membrane receptors that RECRUIT protein kinases, and dimeric protein receptors that ARE protein kinases. And there are other ones as well, but these are the three major ones we are going to talk about. These are the ways that signals get transduced across membranes.
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Seven-Transmembrane-Helix Receptors Change Conformation in Response to Ligand Binding and Activate G Proteins
Seven-transmembrane-helix (7TM) receptors mediate a host of biological functions by responding to a variety of signal molecules (ligands), including hormones, tastants, and even photons.
G protein–coupled receptors are involved in many diseases, and are also the target of nearly half of all modern medicinal drugs.
The binding of a ligand outside the cell induces a structural change in the receptor that can be detected inside the cell.
Created by Brett Barbaro
So these seven-transmembrane-helix receptors (also G-protein-coupled receptors, or just GPCRs) are some of the most important signaling molecules in your body. They respond to hormones, other signaling molecules. Tasting is mediated by G-protein-coupled receptors and sight (vision) - photons can activate them as well. They are involved in many, many diseases, and are the target of about 40% of all drugs that are on the market right now. The general mechanism is that the ligand will bind outside the cell to the seven-transmembrane protein, and that will induce a structural change in the protein that will have an effect inside the cell. It’s a very similar mechanism to allosteric inhibition and several of the other protein activation mechanisms that we’ve talked about.
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Structure of 7TM Receptors
Created by Brett Barbaro
Figure 13.3 The structure of 7TM receptors. (A) Schematic representation of a 7TM receptor showing how it passes through the membrane seven times. (B) Three-dimensional structure of rhodopsin, a 7TM receptor taking part in visual signal transduction. Notice the ligand-binding site near the extracellular surface. As the first 7TM receptor for which the structure was determined, rhodopsin provides a framework for understanding other 7TM receptors. [Drawn from 1F88.pdb.]
This is just a simple diagram. You can see in the upper left there’s seven alpha helices that pass through the membrane, and they bundle together in the confirmation that you see on the right. Now the alpha helices span the membrane, but it’s the hydrophilic residues on the outside of the membrane that are responsible for most of the action of these proteins. There’s a binding site for the ligand, and you can see on the right there it’s buried somewhere in the alpha helix bundle.
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Ligand Binding to 7TM Receptors Leads to the Activation of G Proteins (2/2)
Upon activation by the receptor, the α subunit dissociates from the βγ dimer and exchanges GDP for GTP.
The GTP bound α-subunit transmits the signal to other cellular components.
https://en.wikipedia.org/wiki/Guanosine_triphosphate
GTP
Created by Brett Barbaro
So the seven-transmembrane-receptor protein, or the g-protein-coupled receptor, is associated with a heterotrimer, which means three different molecules coming together to form a complex. And the three different components are the alpha, beta, and gamma subunits of this trimer. In the alpha subunit of the trimer, is a GTPase. We also call that a G-protein. You know, whereas a lot of things use ATP, this protein uses GTP and there’s a diagram of GTP at the bottom. And yeah, it’s the same GTP that you see in your RNA {DNA would be the deoxy form}. So this subunit is bound to, not GTP, but GDP. And the seven-transmembrane receptor interacts with the signal, the ligand, and it undergoes a conformational change, which then changes the conformation of the alpha subunit of the trimer and opens up that active site, which allows the GDP to dissociate from it. And so now you’ve got an open active site, and GDP or GTP can float into there and start interacting. When a GTP comes in, that induces a conformational change, which then causes the alpha subunit to dissociate from the seven-transmembrane receptor, and dissociate from the beta-gamma dimer. So now you’ve got an activated G-protein alpha subunit, and it’s membrane bound, and it’s able to diffuse away from the receptor laterally in the membrane. There are lots of different types of these G-proteins, and the G-protein-coupled receptors and all this, but this action of releasing the GDP and binding a GTP, which then allows it to dissociate and diffuse outward in the membrane, is something that is shared among all of these systems. So it’s kind of like the core mechanism of these G-protein-coupled receptors.
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Activated G Proteins Transmit Signals by Binding to Other Proteins
There are two principal signal transduction pathways involving the G protein–coupled receptors:
the cAMP signal pathway
the phosphatidylinositol signal pathway
The downstream effects of these pathways are highly CELL-TYPE DEPENDENT
Created by Brett Barbaro
What happens after that depends a great deal on the individual case. There {are} two main pathways that get activated by G-protein-coupled receptors. One of them is the cAMP signal pathway, which we have already encountered, and the second is the phosphatidylinositol signal pathway, which we will talk about a little bit later. But even though there are only two main pathways that we are going to talk about, the effects of these pathways are varied a lot. It really depends on what kind of cell it is and the individual environments that these receptors are in.
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Activated G Proteins Transmit Signals by Binding to Other Proteins
In the case of the β-adrenergic receptor signal transduction pathway, the activated G protein, termed Gαs, stimulates the integral membrane enzyme, adenylate cyclase.
Activation of the cyclase leads to the synthesis of the second messenger, cyclic adenosine monophosphate (cAMP).
Created by Brett Barbaro
As far as adrenaline goes, the activated G-protein (the activated alpha subunit) stimulates another integral membrane enzyme called adenylate cyclase, which then leads to the synthesis of cyclic AMP. This is the example that I showed in the beginning.
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Structure of a Heterotrimeric G Protein
Created by Brett Barbaro
Figure 13.5 A heterotrimeric G protein. (A) A ribbon diagram shows the relation between the three subunits. In this complex, the subunit (gray and purple) is bound to GDP. Notice that GDP is bound in a pocket close to the surface at which the a subunit interacts with the βγ dimer. (B) A schematic representation of the heterotrimeric G protein. [Drawn from 1GOT.pdb.]
We are going to dig a little bit deeper into the structure of these G-protein-coupled receptor complexes. The alpha, beta, and gamma complexes you can see in cartoon form on the right. On the left, you can see a more molecular-based representation - ribbons and loops, and you can also see clearly the spot where the GDP and GTP bind in the middle of the protein.
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SIGNAL
RECEPTION
Epinephrine Signal Transduction Pathway
<EDITED by BB>
Created by Brett Barbaro
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Figure 13.4 <EDITED> The activation of protein kinase A by a G-protein pathway. Hormone binding to a 7TM receptor initiates a signal-transduction pathway that acts through a G protein and cAMP to activate protein kinase A.
And so this is just a review of what we saw before. The binding occurs, on the top left, of the adrenaline/epinephrine, and it induces a conformational change in the beta-adrenergic receptor, which then causes the alpha subunit to release the GDP and bind a GTP, and also dissociate from the beta and gamma subunits. And then the GTP-bound alpha subunit will move over and activate the adenylate cyclase.
http://www.rcsb.org/pdb/101/motm.do?momID=58
The adrenaline pathway – David Goodsell illustration
Created by Brett Barbaro
As I’ve mentioned, I think that the previous diagram is pretty poorly drawn. This is a much better diagram, as far as I’m concerned, by David Goodsell. And there is a number of features here that you can see, which are very interesting. First of all, it is much more clear that the alpha, beta and gamma subunits of the G protein are attached to the membrane, and you can see on the top how you have two small hydrophobic tails that become inserted into the membrane. And therefore, this protein is able to diffuse laterally along the membrane, but not to detach from it. Once the alpha subunit has detached and bound to GTP, then it’s able to {diffuse} over to the adenylyl cyclase enzyme, activating it, creating cyclic AMP, and the beta and gamma subunits are wandering free.
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Ligand Binding to 7TM Receptors Leads to the Activation of G Proteins (1/2)
The β-adrenergic receptor is activated by binding to epinephrine, also called adrenaline.
Upon binding of epinephrine, the cytoplasmic aspect to the β-adrenergic receptor activates a heterotrimeric G-protein.
The unactivated G-protein is a heterotrimer consisting of an α subunit, bound to GDP, and β and γ subunits.
Created by Brett Barbaro
This slide is basically just restating a lot of the things that I have just talked about. I am going to say something interesting about epinephrine (or adrenaline). You can see the structure of it on the right. Actually, the words kind of are interesting,. “Epi-” means “upon” in Greek, and “neph” is the Greek root for “kidneys”. “Adrenaline” is basically the Latin form of this word because “ad-” means “on” and “renal” means “kidneys” in Latin. So both of these words are referring to the source of this molecule, which is your adrenal glands, which sit on top of your kidneys.
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Cyclic AMP Stimulates the Phosphorylation of Many Target Proteins by Activating Protein Kinase A
Cyclic AMP activates protein kinase A. Protein kinase A consists of two pairs of subunits, 2 catalytic (C) subunits and 2 regulatory (R) subunits.
Binding of cAMP by the regulatory subunits dissociates these subunits from the complex, resulting in activation of the 2 C subunits.
The activated C subunits continue the epinephrine signal transduction pathway by phosphorylating protein targets that alter physiological functions of the cell.
http://oregonstate.edu/dept/biochem/hhmi/hhmiclasses/bb450/winter2002/molex/camp.htm
Created by Brett Barbaro
A quick look at cyclic adenosine monophosphate, or cAMP - that is what the molecule in the upper left is. It is basically ATP that’s had two phosphates cut off, and then the third phosphate is attached to the ribose ring, forming a new structure. And this structure is very important for activating proteins in the cell - specifically protein kinase A. The cyclic AMP will bind to the regulatory subunits of protein kinase A, and then they will dissociate from the active complex. And then the active subunits will go and do the work.
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Diagram of the Regulation of Protein Kinase A
Created by Brett Barbaro
Figure 13.7 The regulation of protein kinase A. The binding of four molecules of cAMP activates protein kinase A by dissociating the inhibited holoenzyme (R2C2) into a regulatory subunit (R2) and two catalytically active subunits (C).
Here’s a cartoon representation of that process. You can see the cAMP binding domains, there’s four of them. And then the cAMP comes in - but it doesn’t really make a whole lot of sense in this picture.
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http://www.rcsb.org/pdb/101/motm.do?momID=152
Protein kinase A – David Goodsell illustration
Created by Brett Barbaro
Once again, I find the picture by David Goodsell to be much more informative. You can see clearly how the inhibitory molecules, the blue are attached to the pink kinase molecules, and when cyclic AMP binds, it changes the conformation of the inhibitory molecule and releases the active kinase into the cytosol.
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In response to adrenaline:
skeletal muscle cells increase activity digestive muscle cells decrease activity
The downstream effects of cAMP increase are highly CELL-TYPE DEPENDENT
Created by Brett Barbaro
So remember that I had mentioned that the effects of G-protein-coupled receptors are very cell dependent? In response to adrenaline, your muscle cells will increase activity because you need to run and fight - but your digestive muscle cells will actually decrease activity because it is not time to be digesting food, it’s time to be doing other stuff. But it’s the same signal and it’s the same receptor - it’s just how the receptor’s effects inside of the cell are processed that makes the difference.
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http://lsresearch.thomsonreuters.com/maps/2445/
Advanced Biochemistry – a peek behind the curtain
Created by Brett Barbaro
So I just wanted to let you guys know how complicated this stuff is. Of course you are not responsible for this in any way, but I am personally intimidated by this diagram because it is so complicated, all of the downstream effects of adrenaline reception. You can see up at the very top your beta-1 adrenergic receptor, and then right below that is adenylate cyclase and that is as far as we have gone with this, pretty much. You can also see up in the upper left hand corner some ATP and some cAMP. But the downstream effects of all of this are extremely complex, and that is typical of cell signaling cascades.
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G Proteins Spontaneously Reset Themselves Through GTP Hydrolysis
The epinephrine-imitated pathway is shut down in a variety of ways:
Gα has inherent GTPase activity that cleaves the bound GTP to GDP. The Gα bound to GDP spontaneously reassociates with the βγ subunits, terminating the activity of the G protein.
Cyclic AMP phosphodiesterase converts cAMP to AMP, which does not activate protein kinase A.
Epinephrine-β-adrenergic receptor interaction is reversible. Once the concentration of epinephrine falls, the receptor will no longer be active.
Proteins phosphorylated by protein kinase A are dephosphorylated by phosphatases.
Created by Brett Barbaro
The final stage of this signal transduction is the termination stage, and there’s a number of ways that the pathway gets shut down. First of all, the alpha subunit of the GTPase is a GTPase, therefore it breaks down GTP - that is one of its functions - and when it does that, then it becomes inactive. It will start to re-associate with the beta and gamma subunits and also with the seven-transmembrane receptor, and it will stop activating adenylate cyclase. Cyclic AMP is destroyed by a protein called phosphodiesterase, which just hangs around and converts cyclic AMP to AMP wherever it finds it. So cyclic AMP has a short lifetime in the cell - whenever it is turned on it can spread for a bit, it can activate its targets, and then it gets shut off. The third way that it would stop is that you’ve got your epinephrine or adrenaline that is attached to your beta-adrenergic receptor - but it’s not permanently attached; it’s attached with weak bonds and so it can attach and then it can detach. And of course, when it is attached it is functioning and it causes the signal to be transduced, and when it detaches then it stops transducing the signal. So if there’s a high concentration of adrenaline out in the blood, or the extracellular matrix, then you will be activating these receptors very frequently. But once that concentration decreases, all of the adrenaline gets swept away by the blood and the receptors are no longer active. And finally, the proteins that are phosphorylated by protein kinase A, which was activated by the cyclic AMP, are dephosphorylated by phosphatases. That means basically that they’re turned off.
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KINASES add phosphates to specific targets.
PHOSPHATASES remove phosphates from specific targets.
GENERALLY SPEAKING:
KINASES ACTIVATE (turn things on)
PHOSPHATASES DEACTIVATE (turn things off)
Kinases Activate and Phosphatases Deactivate
Created by Brett Barbaro
I think this is a good time to mention that kinases function by adding phosphates to specific targets, and that phosphate (the PO4) is a very important signaling molecule. And oftentimes, when it gets attached to something, then that target will be “turned on”. Phosphatases do the opposite - they detach phosphates from targets; very specific targets, most of the time. So the combination of kinases and phosphatases are responsible for turning on and off broad activities across the cell.
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Diagram of Resetting Gα
Created by Brett Barbaro
Figure 13.8 Resetting G. On hydrolysis of the bound GTP by the intrinsic GTPase activity of Gα, Gα reassociates with the βγ dimer to form the heterotrimeric G protein, thereby terminating the activation of adenylate cyclase.
And this is just a diagram of the termination phase - GTP turns into GDP and re-associates with the beta and gamma subunits.
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Diagram of Signal Termination
Created by Brett Barbaro
Figure 13.9 Signal termination. Signal transduction by the 7TM receptor is halted, in part, by dissociation of the signal molecule (yellow) from the receptor.
And this is a diagram of dissociation of adrenaline from the receptor. Not too much to see here except, that you note that on the left, it’s got a yellow outline and on the right it doesn’t. Well, that is not really what happens. It is a conformational change that takes place in the receptor, and so these two pictures should look slightly different.
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The Hydrolysis of Phosphatidylinositol Bisphosphate by Phospholipase C Generates Two Second Messengers
Some G–protein-coupled receptors activate the phosphoinositide pathway. This pathway involves the Gαq protein as a component of the trimeric G–protein complex.
Gαq activates phospholipase C, which cleaves the membrane lipid phosphatidylinositol bisphosphate into two second messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).
IP3 binds to the IP3-gated channel (IP3 receptor) in the endoplasmic reticulum, allowing an influx of Ca2+ ions into the cytoplasm. The Ca2+ ions regulate a host of cellular functions.
DAG, in conjunction with Ca2+, activates protein kinase C, a serine/threonine kinase.
(GPCR pathway #2)
Created by Brett Barbaro
So we have talked about how G-proteins can activate a phosphorylation cascade, but there is a second pathway that is also very common and that involves the hydrolysis of phosphatidylinositol bisphosphate by phospholipase C. So I mean, that sounds really complicated but basically all it is, is that instead of activating this adenylate cyclase, this G-protein activates another protein called phospholipase C. And in this case, the G-protein, the alpha subunit is called G alpha q (no real particular reason that you need to remember that). So what phospholipase C does is, it takes a particular membrane lipid called phosphatidylinositol bisphosphate, and it cleaves it into two second messengers. One of them is inositol-1,4,5-trisphosphate (or IP3) and the second one is diacylglycerol (DAG), which is still part of the membrane. So basically, it chops off the head of this membrane lipid and sends the water soluble head part out into the cytoplasm, while the remainder stays in the membrane. This IP3 then diffuses out and when it hits this receptor in the endoplasmic reticulum, causes this pore to open up and calcium ions get released from the endoplasmic reticulum into the cell. And the calcium ions have a whole bunch of other downstream effects. Calcium in general, actually, is a signal for cells to be ON, more or less - it’s “something is happening - there’s calcium around, so time to do something!” There are a number of different functions. Like I said, these signal transduction pathways are extraordinarily complex. And to add another layer of complexity to it, the diacylglycerol that is still stuck in the membrane does something else as well. It activates protein kinase C, which is a kinase which will phosphorylate its targets. And that happens to be a serine/threonine kinase, so it phosphorylates on a serine or threonine side chain. Incidentally, you’ll notice that protein kinase A is activated by cyclic AMP, and protein kinase C is activated by calcium, which starts with a “C”, and I actually think that is where they got the names from. So it wasn’t like there was an A, B, and C, and all of that. I think they named it A because of the cyclic AMP and C because of the calcium.
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The Phospholipase C Reaction
Created by Brett Barbaro
Figure 13.11 The phospholipase C reaction. Phospholipase C cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate into two second messengers: diacylglycerol, which remains in the membrane, and inositol 1,4,5-trisphosphate, which diffuses away from the membrane.
So you see here a diagram of phosphatidylinositol 4,5-bisphosphate (PIP2). And that’s the phosphatidate on the left, and then the inositol with all of the phosphates on it to the right, and the glycerol backbone there in the middle. And when it gets in contact with the phospholipase-C, it gets cut into two pieces - the left hand one, the diacylglycerol, stays in the membrane, and the one on the right, inositol 1,4,5-trisphosphate (IP3), diffuses out into the cytoplasm - but they’re both important for the signaling.
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Diagram of the Phosphoinositide Cascade
Created by Brett Barbaro
Figure 13.12 The phosphoinositide cascade. The cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) results in the release of calcium ions (owing to the opening of the IP3 receptor ion channels) and the activation of protein kinase C (owing to the binding of protein kinase C to free DAG in the membrane). Calcium ions bind to protein kinase C and help facilitate its activation.
So here’s an example/diagram of how this all works. At the top, we can see PIP2 (which is your phosphatidylinositol 4,5-bisphosphate) sticking out of the membrane. And phospholipase C, which is activated by a G-protein-coupled receptor, will cleave the IP3 off of that, which will then float down into the IP3 receptor there on the endoplasmic reticulum, and that will cause a conformational change which will allow calcium ions to escape into the cytoplasm. Once they escape into the cytoplasm, they interact with protein kinase C (you can see the blue large molecule up on the right, and the two calciums that are attached to it) - but protein kinase C also needs to interact with diacylglycerol to be activated.
Now, you might ask, “Why? Why all of this complicated stuff?” Well, it’s not easy to answer that question. These systems are very complex and they control a large number of things within the cell - but I will tell you one thing is that when you have a number of steps like this that are involved in getting a signal to be transduced, then you have a number of opportunities at each one of those points to alter the signal. So the very fact that this signal is complex means it is also very sensitive and can be regulated in many different ways. And that’s probably one of the reasons that it has become one of the central signal transduction pathways in cells.
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Section 13.3 Some Receptors Dimerize in Response to Ligand Binding and Recruit Tyrosine Kinases
Learning objective 5: Differentiate the various types of membrane receptors.
There are three major classes of membrane receptors:
Seven-transmembrane-helix receptors associated with heterotrimeric G-proteins.
Dimeric membrane receptors that recruit protein kinases.
Dimeric protein receptors that are protein kinases.
Created by Brett Barbaro
So that’s enough about the G-protein-coupled receptors. We’re going to move onto dimeric membrane receptors that recruit protein kinases right now.
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Section 13.3 Some Receptors Dimerize in Response to Ligand Binding and Recruit Tyrosine Kinases
Receptor dimerization may result in tyrosine kinase recruitment
Human growth hormone receptor is a monomeric integral membrane protein with an extracellular and intracellular domain joined by an intramembrane α helix.
Upon hormone binding, the receptor dimerizes.
DID YOU KNOW?
Overstimulation of the growth-hormone signal-transduction pathway can lead to pathological conditions. Acromegaly is a rare hormonal disorder resulting from the overproduction of growth hormone in middle age. A common characteristic of acromegaly is enlargement of the face, hands, and feet. Excessive production of growth hormone in children results in gigantism. André the Giant, who played the beloved giant Fezzik in Rob Reiner’s classic film The Princess Bride suffered from gigantism.
Created by Brett Barbaro
...and remember that kinases attach phosphates to things and turn them on, basically. So for the example that we’re going to use on this one is the human growth hormone receptor. It’s normally just a monomeric integral membrane protein that floats around with a domain on the outside and a domain on the inside, and a single alpha helix combine them. Once the human growth hormone gets bound to the extracellular domain, then it causes the receptor to dimerize. It’s kind of an interesting point, is that since you only have one alpha helix that goes through the membrane, it’s pretty hard to induce a conformational change in that alpha helix that can be detected on the other side of the membrane. There’s really not a lot that you can change in the conformation. So this other method is a way that you can have a single helix, a very simple length of protein going across the membrane, and still be able to transduce a signal across the membrane.
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Structure of the Binding of a Growth Hormone Leading to Receptor Dimerization
Created by Brett Barbaro
Figure 13.13 The binding of growth hormone leads to receptor dimerization. (A) A single growth-hormone molecule (blue) interacts with the extracellular domains of two receptors (red). (B) The binding of one hormone molecule to two receptors leads to the formation of a receptor dimer. Dimerization is a key step in this signal-transduction pathway.
And this is a diagram showing that, with two red extracellular domains that are interacting with the blue growth hormone up at the top.
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Diagram of Receptor Dimerization from the Binding of a Growth Hormone
Created by Brett Barbaro
You can see on the left here the extracellular domains and intracellular domains floating around independent of each other. {I mean that the individual transmembrane proteins are floating around independently – the domains are always tied to each other.} When the growth hormone binds, then they become dimerized and they turn yellow. No, they don’t really turn yellow. But they do start to interact with each other, and that’s what causes the signal to be transduced.
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Receptor Dimerization May Result in Tyrosine Kinase Recruitment
Dimerization of the extracellular domains of the receptor brings together the intracellular domains, which are associated with Janus kinase 2 (JAK2).
Each JAK phosphorylates its partner on a tyrosine residue, activating the two kinases.
The activated kinases then phosphorylate other targets, including a regulator of gene expression called signal transducer and activator of transcription 5 (STAT5). STAT5 further propagates the signal by altering gene expression.
Created by Brett Barbaro
So we’re going to go into some more detail on this system, and it’s not because you need to know all of these details, but it’s to get an idea of how this overall process would work. This is a very important, common process, so it’s a good idea to become familiar with it. What happens in the case of these particular growth hormone receptors is, they are dimerized and the intracellular domains are associated with another protein called Janus kinase 2, or JAK2. These JAKs, when they get close together, phosphorylate tyrosine residues on the other molecule. And this is perhaps the most important thing to learn from this example - that tyrosines are often phosphorylated. Serines and threonines can be phosphorylated as well, but when we’re talking about signal cascades, tyrosine kinases are actually some of the most important kinases that are involved in cell signaling. Activated kinases then phosphorylate other targets, including a regulator of gene expression - STAT5. And that goes down into the nucleus and alters gene expression.
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Diagram of the Cross-phosphorylation of Two Molecules of JAK2 Induced by Receptor Dimerization
Created by Brett Barbaro
Figure 13.14 The cross-phosphorylation of two molecules of JAK2 induced by receptor dimerization. The binding of growth hormone (blue) leads to growth-hormone-receptor dimerization, which brings two molecules of Janus kinase 2 (JAK2, yellow) together in such a way that each phosphorylates key residues on the other. The activated JAK2 molecules remain bound to the receptor.
This is a terrible diagram that’s in your book, so I suggest that you ignore it...
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https://www.studyblue.com/notes/note/n/10-2-12-830am-molec-cell-signaling-ji/deck/3894855
See also: http://media.cellsignal.com/www/html/science/landscapes/rtk/rtk.html#MMP-2
JAK-STAT Receptor Signaling
Created by Brett Barbaro
...and look at this diagram instead. You can clearly see, on the left here, the trans-membrane proteins have already been dimerized, and they’re already associated with these JAK proteins. These JAK proteins then phosphorylate each other and they phosphorylate the receptor once again on tyrosine residues. Then the STATs are able to associate with the phosphorylated tyrosines, and then the JAK kinases phosphorylate the STATs. The phosphorylated STATs can then associate with each other and dimerize where they translocate to the nucleus. Once again, I have to apologize that the whole system is so complicated that ... I can’t grasp it in my own brain all at once. But these are important examples of how this might work, and if you become familiar with this pathway, then you will know something more about generalized pathways throughout the cell. When you’re talking about an individual pathway, a specific pathway, you’re best off looking that up. Not many people actually know pathways by memory. Plus, the state of our knowledge of these pathways is constantly changing and there’s new stuff being discovered, so it’s not even certain that the most up to date information is correct. I think the overall idea here is that something comes from the outside, causes something in the membrane to change, and then that causes something inside the cell to change, and then ends up with something diffusing away from the membrane carrying a signal.
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Section 13.2 Receptor Proteins Transmit Information into the Cell
Learning objective 5: Differentiate the various types of membrane receptors.
There are three major classes of membrane receptors:
Seven-transmembrane-helix receptors associated with heterotrimeric G-proteins.
Dimeric membrane receptors that recruit protein kinases.
Dimeric protein receptors that are protein kinases.
Created by Brett Barbaro
All right, and then we have dimeric protein receptors that ARE protein kinases. I don’t know why this is particularly, you know different than things that recruit protein kinases - in my mind they are all kind of the same - but this will give us a chance to talk about a couple of more interesting things.
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Clinical Insight: Some Receptors Contain Tyrosine Kinase Domains Within Their Covalent Structures
Some growth factors and hormone receptors, such as the epidermal growth factor (EGF) and insulin, bind to receptors that are tyrosine kinases, called receptor tyrosine kinases (RTK). Upon growth factor or hormone binding, these receptors form dimers.
Receptor dimerization leads to cross-phosphorylation and activation of the two intracellular kinase domains.
The phosphorylated kinases form docking platforms for other components of the signal transduction pathway.
Mutations in these receptors in humans cause a variety of pathologies.
Created by Brett Barbaro
Interesting things such as epidermal growth factor and insulin! I’m sure you’re all familiar with insulin and its role in diabetes. Several of you have asked to go into more detail, well, here is some molecular detail about it. Both of these things, EGF and insulin, bind to receptors on the outside of the cells, and these receptors are tyrosine kinases. The tyrosine kinase domains are on the inside of the cell, and when the hormone (the growth factor or hormone) binds to the domain on the outside of the cell, it causes the receptors to form dimers {this is generally true, but insulin receptors are an exception - they are always dimers, and binding causes a conformational change}. And then the inner parts can interact and phosphorylate each other and induce conformational changes, which then have downstream effects.
I think it’s important to remember that these receptors are floating around all the time, even when they’re not attached to anything - and I think they can probably bump into each other and phosphorylate each other at a low level, just randomly, without interacting with any kind of signal. But there’s nothing to hold them together - so after they might interact briefly, they drift apart and revert to their original state. There might be some baseline activity due to this, but to get a really strong signal you need to have them dimerized.
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The modular structure of the EGF receptor
Created by Brett Barbaro
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The modular structure of the EGF receptor. This schematic view of the amino acid sequence of the EGF receptor shows the EGF-binding domain that lies outside the cell, a single transmembrane helix-forming region, the intracellular tyrosine kinase domain, and the tyrosine-rich domain at the carboxyl terminus.
So this is a diagram of the EGF receptor - and this type of diagram shows basically the linear amino-acid sequence. On the left, in yellow, the first part of the sequence is forming the EGF-binding domain outside of the cell. The next little white bit is a transmembrane helix, which will turn into an alpha helix with a lot of hydrophobic residues on it. And then inside you have the kinase domain, and then the C-terminal tail, which is tyrosine rich - and you could guess that is where all of the tyrosines get phosphorylated.
Diagram of the EGF Signaling Pathway
Created by Brett Barbaro
Figure 13.15 The EGF signaling pathway. The binding of epidermal growth factor (EGF) to its receptor leads to cross-phosphorylation of the receptor. The phosphorylated receptor binds Grb-2, which, in turn, binds Sos. Sos stimulates the exchange of GTP for GDP in Ras. Activated Ras binds to protein kinases and stimulates them (not shown).
So in this diagram we see how the EGF gets bound to the extracellular domains of the receptor, bringing them in proximity to each other, so they can phosphorylate each other in the intracellular domain. And then, after the phosphorylation takes place, this GRB-2 protein, and its other associated proteins, are able to {exchange} the GDP {for} a GTP on this Ras protein. And this Ras protein is actually very important for cancer. There’s a very well known pathway called “Ras-Raf-MEK-ERK” {or MAPK/ERK}, and things get phosphorylated twice, and it’s just crazy in there - but in general it all gets started by the EGF coming to the surface and causing dimerization of these receptor tyrosine kinases.
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Ras Belongs to Another Class of Signaling G Proteins
A key component of the EGF pathway, as well as other signal-transduction pathways, is the protein Ras.
Ras is a member of the family of signal proteins called small G proteins or small GTPases. The small G proteins are monomeric.
Members of this family control a variety of cellular processes.
Like the Gα protein, Ras is active when bound to GTP and inactive when bound to GDP. Ras also has intrinsic GTPase activity, which controls signal duration.
Other proteins function to modulate the GTPase activity of Ras.
Created by Brett Barbaro
Now Ras, actually, is not a G-protein-coupled receptor - it’s not like an alpha subunit of your G-proteins - but it’s very similar to the alpha subunit because it binds GTP and then hydrolyzes it to GDP. When it’s bound to GTP, then it is active - and when it hydrolyzes the GTP, it becomes inactive. So it has a short, kind of, signal duration. Once you’ve attached the GTP to it, then it will go around and do some stuff for a little while, but it will eventually hydrolyze the GTP and become inactive.
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Section 13.4 Metabolism in Context: Insulin Signaling Regulates Metabolism
The polypeptide hormone insulin is secreted when the blood is rich in glucose.
Insulin is the biochemical signal for the fed state.
Created by Brett Barbaro
Now insulin, insulin is secreted by the pancreas when the blood is rich in glucose. So there are some cells in the pancreas that are able to detect the level of glucose in the blood, and then they are able to secrete the insulin, and that insulin circulates through your body and tells your cells that it’s time to eat. So the cells will then start absorbing more glucose from the blood into the internal parts of the cell.
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The Insulin Receptor Is a Dimer That Closes Around a Bound Insulin Molecule
Insulin consists of two polypeptide chains linked by disulfide bonds.
The insulin receptor is a receptor tyrosine kinase.
Unlike the other members of the receptor tyrosine kinase class, the insulin receptor exists as a dimer even in the absence of insulin.
Insulin binding causes a change in quaternary structure, leading to cross-phosphorylation and activation of the kinase domains.
Created by Brett Barbaro
We talked earlier about insulin, how it’s two chains actually linked by disulfide bonds. And, of course, we just mentioned that its receptor is a tyrosine kinase, which starts to cross-phosphorylate.
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http://www.rcsb.org/pdb/101/motm.do?momID=14
Figure 13.17 Insulin structure
Created by Brett Barbaro
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Figure 13.16 Insulin structure. Notice that insulin consists of two chains (shown in blue and yellow) linked by two interchain disulfide bonds. The chain (blue) also has an intrachain disulfide bond. [Drawn from IB2F.pdb.]
This is a good picture of insulin, actually. The picture that we had seen before looked like two straight lines combined with some other straight lines. Well, it’s a little more complicated than that. What you can see in this big diagram is all of the atoms (this is a stick diagram), and you can see the disulfide bridges - they are numbered with a green 1, 2, and 3 - and then the hydrophobic core of the protein - remember, it is a protein, and when it folds up, the hydrophobic residues tend to be on the inside of the protein, and the charged residues are on the surface. So on the left you can see some other representations, the ribbon diagram and the space filling diagrams, which will give you an idea of what the overall shape of the insulin molecule is.
Diagram of the Insulin Receptor
http://www.rcsb.org/pdb/101/motm.do?momID=182
Created by Brett Barbaro
Figure 13.17 The insulin receptor. The receptor consists of two units, each of which consists of an α subunit and a β subunit linked by a disulfide bond. The α subunit lies outside the cell, and two α subunits come together to form a binding site for insulin. Each β subunit lies primarily inside the cell and includes a protein kinase domain.
So on the left here, we have a picture of the insulin receptor. This is a cartoon picture - not very accurate I would have to say. On the right, we have the actual structure of the insulin receptor. It’s actually like two hooks that come together, and the insulin binds to the outside of the hooks - the insulin is that little orange bit on the outside there - and then the beta subunits, on the inside, are the ones that phosphorylate each other.
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The Activated Insulin-Receptor Kinase Initiates a Kinase Cascade
Insulin binding causes a change in the quaternary structure that results in cross-phosphorylation by the two kinase domains, activating the kinase. The activated kinase of the insulin receptor phosphorylates insulin-receptor substrates (IRSs).
The phosphorylated IRSs are adaptor proteins to convey the insulin signal.
Phosphoinositide-3 kinase binds IRS and then phosphorylates phosphatidylinositol 4,5-bisphophate (PIP2) to form phosphatidylinositol 3,4,5-trisphosphate (PIP3).
PIP3 activates PIP3-dependent kinase, which in turn, phosphorylates and activates the kinase Akt.
Akt phosphorylates enzymes that control the trafficking of the glucose transporter (GLUT4), increasing glucose uptake by the cells, as well a enzymes that convert glucose into glycogen.
Created by Brett Barbaro
So - another mess of a kinase cascade for you! It starts off with the insulin receptor phosphorylating insulin-receptor substrates (extremely creative name {not}). These substrates then convey the signal to other proteins inside the cell. One of these is the phosphoinositide-3 kinase. Now, I think you can guess what this does. It takes phosphoinositide and adds another phosphate group to it, changing a PIP2 (you recall PIP2 from a few slides ago) into PIP3. This PIP3 is going to be membrane bound with inositol and some phosphates on it, and interact with a different protein called PIP3 dependent kinase, which then phosphorylates and activates a kinase called AKT. This AKT phosphorylates the glucose transporter, and eventually leads to an increase in glucose uptake by cells, in addition to also some downstream activity, which increases glycogen production - which would make sense, because you have extra glucose, then you can store some extra glycogen.
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Diagram of Insulin Signaling
Created by Brett Barbaro
Figure 13.18 Insulin signaling. The binding of insulin results in the cross-phosphorylation and activation of the insulin receptor. Phosphorylated sites on the receptor act as binding sites for insulin-receptor substrates such as IRS-1. The lipid kinase phosphoinositide 3-kinase binds to phosphorylated sites on IRS-1 through its regulatory domain and then converts PIP2 into PIP3. Binding to PIP3 activates PIP3-dependent protein kinase (PDK1), which phosphorylates and activates kinases such as Akt. Activated Akt can then diffuse throughout the cell to continue the signal-transduction pathway.
Here’s a cartoon diagram of that, what I just told you. The insulin combines with the insulin receptors to {activate} these tyrosine kinases, which then phosphorylate each other, interact with this IRS, which is phosphorylated; that interacts with phosphoinositide-3 kinase, which then interacts with PIP2, turning it into PIP3, and then the PIP3 will involve PDK1 and that PDK1 will phosphorylate AKT, which will then go on and do a number of things. Once again - you’re not required to know all of these different things, but if I show you a diagram of this process, I would expect that you would be able to at least trace it and understand more or less what’s happening: how the signal is getting from outside the cell, to the inside of the cell.
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The Action of a Lipid Kinase in Insulin Signaling
Created by Brett Barbaro
Figure 13.19 The action of a lipid kinase in insulin signaling. Phosphorylated IRS-1 and IRS-2 activate the enzyme phosphatidylinositide 3-kinase, an enzyme that converts PIP2 into PIP3.
Here is a close up on the phosphatidylinositol 4,5-bisphosphate transformation. It very simply just adds a phosphate group to one of the hydroxide residues.
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Diagram of the Insulin Signaling Pathway
Created by Brett Barbaro
Figure 13.20 Insulin signaling pathway. Key steps in the signal-transduction pathway initiated by the binding of insulin to the insulin receptor.
So here is just an overall view of that whole process from insulin down to Akt, and you’ll note that there are actually three steps in this process which are merely amplifying the signal.
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Insulin Receptor Signaling
Created by Brett Barbaro
Here is a diagram that is a little bit more complete as far as what’s going on in the cell. Once again, I am not going to hold you responsible for any of this. But you can see at the top the insulin receptor, and the IRS is attached to it on the interior side. Trace that to the PI3K and PIP3 (which you can see there), and then Akt2. And if you look at the left hand side you’ll see a circular object called the GLUT4 vesicle, and that is a small membrane-bound compartment that has the glucose transporter in it. And that is contained within the cell - and what happens when you get the insulin is that it will cause that vesicle to go up to the surface of the cell and fuse with the cell’s lipid membrane, and therefore introduce more of these glucose transporters and therefore increase the glucose uptake. And then, you can see at the bottom also there’s stuff happening in the nucleus of the cell.
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Insulin Signaling Is Terminated by the Action of Phosphatases
Protein phosphatases remove phosphates from the activated proteins in the insulin signal-transduction pathway, terminating the insulin signal.
Lipid phosphatases contribute to signal termination by converting PIP3 into PIP2.
Created by Brett Barbaro
Most of these steps involve the phosphorylation of substrates. So to turn them off, you use phosphatases to remove the phosphates from these substrates. There is a lipid phosphatase which removes phosphates from lipids, and that will convert the PIP3 back into PIP2.
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13.5 Calcium Ion Is a Ubiquitous Cytoplasmic Messenger
Ca2+ is an important second messenger in eukaryotic signal transduction pathways.
It is a critical component of muscle contraction and neurotransmitter release.
Recall that the Ca2+ also plays a role in the phosphoinositide cascade.
Created by Brett Barbaro
Just one quick mention here, calcium is an important second messenger that’s in a lot of signal transduction pathways, some of which are muscle contraction and neurotransmitter release {very important stuff!}. Remember also that the calcium plays a role in the phosphoinositide cascade.
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