1 00:00:15 --> 00:00:18 We're going to continue our discussion today about cell biology, 2 00:00:18 --> 00:00:21 and specifically about the subject of signalling which we talked about 3 00:00:21 --> 00:00:24 in general terms last time. I gave you some of the general 4 00:00:24 --> 00:00:28 principles about signaling, signal propagation and signal 5 00:00:28 --> 00:00:32 processing in biological systems. And today we're going to talk about 6 00:00:32 --> 00:00:36 two specific examples. First a short-range response which 7 00:00:36 --> 00:00:41 results in the production of second messenger cyclic AMP and the results 8 00:00:41 --> 00:00:46 of that in terms of downstream events. And the second will be an 9 00:00:46 --> 00:00:50 example of a long-range response which requires new gene expression, 10 00:00:50 --> 00:00:55 specifically in the stimulation of cells to divide. 11 00:00:55 --> 00:01:00 And the example I'll give you in the context of wound healing. 12 00:01:00 --> 00:01:08 So just to finish up what I started at the end of last lecture. 13 00:01:08 --> 00:01:18 As I mentioned, 14 00:01:18 --> 00:01:21 the energy associated with the binding of a single ligand to a 15 00:01:21 --> 00:01:25 single receptor molecule at the cell's surface is insufficient to 16 00:01:25 --> 00:01:28 trigger any of the downstream events that are necessary either for the 17 00:01:28 --> 00:01:32 short-term or long-term responses. And so it's necessary that the 18 00:01:32 --> 00:01:36 signal be amplified. And there are various ways that 19 00:01:36 --> 00:01:40 this happens. The first, which I mentioned last time, 20 00:01:40 --> 00:01:44 is the production of molecules which we refer to as second messengers. 21 00:01:44 --> 00:01:48 Second because they are not the primary thing. 22 00:01:48 --> 00:01:52 The primary thing is the ligand binding to the receptor. 23 00:01:52 --> 00:01:56 They come after that messenger because they transmit the message 24 00:01:56 --> 00:02:00 related to the binding of the ligand to the receptor inside the cell. 25 00:02:00 --> 00:02:04 And the example that I mentioned and that we'll talk about in more 26 00:02:04 --> 00:02:08 detail today is a second messenger called cyclic AMP or cAMP. 27 00:02:08 --> 00:02:12 I also mentioned that there are examples of enzymatic cascades. 28 00:02:12 --> 00:02:16 And, again, the purpose here is to amplify the signal. 29 00:02:16 --> 00:02:20 One enzyme turns on another enzyme which turns on many more enzymes, 30 00:02:20 --> 00:02:24 et cetera. It's like a pyramid effect where something little grows 31 00:02:24 --> 00:02:28 to something big. And the example I gave you last 32 00:02:28 --> 00:02:33 time was the analogy to dominos. Pushing one over basically spreads 33 00:02:33 --> 00:02:39 the signal down the chain. So signal amplification with 34 00:02:39 --> 00:02:45 respect to second messengers, the principle is quite simple. 35 00:02:45 --> 00:02:51 Imagine an enzyme which is in an inactive state. 36 00:02:51 --> 00:02:57 The binding of ligand to receptor makes that enzyme go 37 00:02:57 --> 00:03:03 to an active state. And in its active state it can 38 00:03:03 --> 00:03:09 convert a substrate into a product. Now, if that product can stimulate 39 00:03:09 --> 00:03:16 a second enzyme, imagine enzyme two, 40 00:03:16 --> 00:03:23 which is in an inactive state now in the presence of this product, 41 00:03:23 --> 00:03:30 is converted to an active state, you've amplified the signal. 42 00:03:30 --> 00:03:35 You've turned on a lot of enzymes which make even more of this product 43 00:03:35 --> 00:03:41 which now stimulate yet another enzyme. So this process then 44 00:03:41 --> 00:03:46 results in a form of signal amplification. 45 00:03:46 --> 00:03:52 The same is true for enzyme cascades. If you imagine an enzyme 46 00:03:52 --> 00:03:57 which is in an inactive state. The binding of a ligand to a 47 00:03:57 --> 00:04:03 receptor converts that enzyme to an active state. 48 00:04:03 --> 00:04:08 If that enzyme now acts on another enzyme, E2 goes from inactive state 49 00:04:08 --> 00:04:14 to an active state, and that enzyme then acts on another 50 00:04:14 --> 00:04:19 enzyme, E3 goes from an inactive state to an active state, 51 00:04:19 --> 00:04:25 again, you've gone from a relatively small amount of stimulus propagated 52 00:04:25 --> 00:04:30 through the stimulation of this enzymatic activity, 53 00:04:30 --> 00:04:36 which acts on a number of these to make even more of these active, 54 00:04:36 --> 00:04:41 which acts on even more of these to make even more of these active, 55 00:04:41 --> 00:04:47 again, one develops signal amplification. OK? 56 00:04:47 --> 00:04:58 Now, these changes that take place, 57 00:04:58 --> 00:05:02 both in the generation of second messenger, as well as in the turning 58 00:05:02 --> 00:05:06 on of enzymes, often result from changes in protein 59 00:05:06 --> 00:05:10 states. I tried to give you indications last time that proteins 60 00:05:10 --> 00:05:14 are not static things. They can move, they can interact, 61 00:05:14 --> 00:05:18 and they can actually change shape. And changes in protein states are 62 00:05:18 --> 00:05:22 often reflected in changes in protein structure, 63 00:05:22 --> 00:05:26 subtle changes in protein structure, but nevertheless changes in protein 64 00:05:26 --> 00:05:31 structure. And these can come in noncovalent 65 00:05:31 --> 00:05:36 forms. The protein is not changed by any new covalent bonds but by 66 00:05:36 --> 00:05:41 binding to something in a noncovalent interaction. 67 00:05:41 --> 00:05:47 And the classic example of this are G-binding proteins. 68 00:05:47 --> 00:05:57 These proteins bind guanine 69 00:05:57 --> 00:06:03 nucleotides. That's why they're called G-binding proteins. 70 00:06:03 --> 00:06:09 And they're often shortened and referred to as G proteins. 71 00:06:09 --> 00:06:15 We'll meet two G proteins later in today's lecture. 72 00:06:15 --> 00:06:21 G proteins change their structure depending on whether or not they're 73 00:06:21 --> 00:06:27 bound to GTP or GDP. Typically, in their inactive state, 74 00:06:27 --> 00:06:33 they're bound to GDP. However, in response to a signal transduction 75 00:06:33 --> 00:06:39 pathway, there's an exchange event whereby GTP binds to the molecule 76 00:06:39 --> 00:06:46 and GDP is released. This reaction is usually catalyzed 77 00:06:46 --> 00:06:54 by another protein known as a nucleotide exchange factor. 78 00:06:54 --> 00:07:02 We're exchanging this nucleotide for this nucleotide, 79 00:07:02 --> 00:07:10 and in so doing we change the shape of the molecule. 80 00:07:10 --> 00:07:15 When it's bound to GTP now it has a different shape. 81 00:07:15 --> 00:07:20 And by virtue of this different shape, it might now be able to 82 00:07:20 --> 00:07:25 interact preferentially with another molecule. So imagine that we have a 83 00:07:25 --> 00:07:31 signaling molecule which has a shape that looks something like this. 84 00:07:31 --> 00:07:38 A signaling protein. 85 00:07:38 --> 00:07:43 Given the lack of complementarity between this shape and this shape, 86 00:07:43 --> 00:07:48 these proteins will not interact. However, upon nucleotide exchange 87 00:07:48 --> 00:07:53 such that the protein now binds GTP and now changes its shape, 88 00:07:53 --> 00:07:58 now we can get a productive interaction between the G protein 89 00:07:58 --> 00:08:03 and the signaling molecule. And this can lead to a form of 90 00:08:03 --> 00:08:08 signal amplification. OK? So that's an example of a 91 00:08:08 --> 00:08:14 noncovalent change, and a reversible one. 92 00:08:14 --> 00:08:24 The GTP can be hydrolyzed -- 93 00:08:24 --> 00:08:30 -- back to GDP, thereby shutting the signal off. 94 00:08:30 --> 00:08:36 OK? Another example of a structural change in a protein but 95 00:08:36 --> 00:08:49 now a covalent one -- 96 00:08:49 --> 00:08:56 -- is in the form of phosphorylation. So imagine that we have a protein 97 00:08:56 --> 00:09:03 here, an enzyme which is in its inactive state. 98 00:09:03 --> 00:09:07 And this protein has among its amino acids, in some critical position, 99 00:09:07 --> 00:09:12 an amino acid with a hydroxyl residue. Does anybody remember back 100 00:09:12 --> 00:09:17 in the Dark Ages when you were learning your amino acid structures 101 00:09:17 --> 00:09:22 which amino acids have hydroxyl residues? I'm tempted to offer 102 00:09:22 --> 00:09:27 money to anybody who would remember all three. Anybody. 103 00:09:27 --> 00:09:33 Does anybody remember even one? Serine it is. Yes, 104 00:09:33 --> 00:09:39 that's one. Serine. Anything else? Tyrosine is another. 105 00:09:39 --> 00:09:45 Very good. And the last one? Threonine. So these three amino 106 00:09:45 --> 00:09:52 acids have hydroxyl residues. And by virtue of that they are 107 00:09:52 --> 00:09:58 subject to, or can be subject to protein phosphorylation at the hands 108 00:09:58 --> 00:10:04 of enzymes, which I've referred to in previous lectures, called 109 00:10:04 --> 00:10:10 protein kinases. These proteins can be modified such 110 00:10:10 --> 00:10:15 that they have a phosphate group on them. And this can change the 111 00:10:15 --> 00:10:19 structure of the protein and make it go from an inactive to an active 112 00:10:19 --> 00:10:24 state. There are actually examples where it goes the other way, 113 00:10:24 --> 00:10:29 phosphorylation takes an active protein and makes it inactive, 114 00:10:29 --> 00:10:35 but you see the point. Phosphorylation can change the 115 00:10:35 --> 00:10:41 activity. And, once again, this is reversible. 116 00:10:41 --> 00:10:47 There are protein phosphatases which will remove the phosphate and 117 00:10:47 --> 00:10:53 return the protein to its baseline state. OK? So just to give you an 118 00:10:53 --> 00:10:59 example of how this might be useful. 119 00:10:59 --> 00:11:05 Imagine that I have an enzyme here which has an active site, 120 00:11:05 --> 00:11:12 but this active site, by virtue of the confirmation of the protein, 121 00:11:12 --> 00:11:19 is closed. That is the substrates cannot get in there in order to be 122 00:11:19 --> 00:11:25 modified. At the hands of a specific kinase that might recognize 123 00:11:25 --> 00:11:32 a hydroxyl residue right here, the shape of this protein might 124 00:11:32 --> 00:11:38 change. So now the active site becomes open 125 00:11:38 --> 00:11:44 and the enzyme becomes active. OK? And likewise this can be 126 00:11:44 --> 00:11:49 reversed through the action of a phosphatase. OK? 127 00:11:49 --> 00:11:55 So protein structures change, activities of proteins, enzymes 128 00:11:55 --> 00:12:01 change. And that's one way in which we can propagate signals. 129 00:12:01 --> 00:12:05 OK. So let's now go to a specific example. And the example that I 130 00:12:05 --> 00:12:09 want to give you is one that hopefully not many of you are 131 00:12:09 --> 00:12:13 familiar with. If you're camping and you run 132 00:12:13 --> 00:12:17 across this fellow here, what is your immediate reaction? 133 00:12:17 --> 00:12:21 You run. You run. You never try to feed the bear a marshmallow with 134 00:12:21 --> 00:12:25 your mouth. That's the first thing I learned about camping, 135 00:12:25 --> 00:12:30 you run. This is the famous fight or flight response. 136 00:12:30 --> 00:12:36 And when you're dealing with a bear, you want to take the flight option. 137 00:12:36 --> 00:12:42 It's stimulated by fear, some perception of fear. And upon 138 00:12:42 --> 00:12:59 perception of fear -- 139 00:12:59 --> 00:13:02 -- through a complex physiology, a small molecule is released from 140 00:13:02 --> 00:13:06 your adrenal glands. Glands that sit on top of your 141 00:13:06 --> 00:13:10 kidneys. This molecule is called epinephrine, otherwise known as 142 00:13:10 --> 00:13:14 adrenaline. So this is released from your adrenal glands and various 143 00:13:14 --> 00:13:17 things happen. One, that we're going to spend a 144 00:13:17 --> 00:13:21 little time talking about, is that your blood glucose levels go 145 00:13:21 --> 00:13:25 up. Why? Why do you want your blood glucose levels to go up when 146 00:13:25 --> 00:13:29 you see this fellow here? So you can run. 147 00:13:29 --> 00:13:33 Remember, that's the first thing you want to do when you see a bear, 148 00:13:33 --> 00:13:37 run. In addition, your heart rate goes up so you can pump more blood 149 00:13:37 --> 00:13:42 to your big muscles, your breathing rate goes up so you 150 00:13:42 --> 00:13:46 can oxygenate those muscles, and your blood pressure goes up. 151 00:13:46 --> 00:13:51 And there are other things that happen as well, 152 00:13:51 --> 00:13:55 but these are the most important with respect to the fight 153 00:13:55 --> 00:14:00 and flight response. This happens because this hormone, 154 00:14:00 --> 00:14:05 epinephrine, triggers all of these responses, actually in different 155 00:14:05 --> 00:14:10 cells within your body. Because it's acting at distance 156 00:14:10 --> 00:14:15 sites within your body, from the adrenal glands to the 157 00:14:15 --> 00:14:20 muscles to the liver to the heart to your brain, this is referred to as 158 00:14:20 --> 00:14:25 an endocrine response. An endocrine response is basically 159 00:14:25 --> 00:14:30 defined as hormones acting at a distance. 160 00:14:30 --> 00:14:41 OK? Produced somewhere. 161 00:14:41 --> 00:14:50 Acting somewhere else in your body. We differentiate that with the 162 00:14:50 --> 00:15:00 paracrine response in which there is a local release of growth factors 163 00:15:00 --> 00:15:10 and other ligands. 164 00:15:10 --> 00:15:15 So here there's a local issue. Something happens locally, factors 165 00:15:15 --> 00:15:20 are released right in the environment, and they act upon cells 166 00:15:20 --> 00:15:26 right in their neighborhood. And finally a related turn called 167 00:15:26 --> 00:15:41 autocrine which is cells -- 168 00:15:41 --> 00:15:44 -- which release the factor and bind it themselves. 169 00:15:44 --> 00:15:47 Autocrine. So they stimulate themselves. And very often you'll 170 00:15:47 --> 00:15:51 start a process one of these ways. And then the cell will take over. 171 00:15:51 --> 00:15:54 Basically amplifying the signal for itself. OK? Now, 172 00:15:54 --> 00:15:58 in the example that I want to talk about in the first portion 173 00:15:58 --> 00:16:02 of the lecture. We're going to deal with this one 174 00:16:02 --> 00:16:08 here, blood glucose, the increase levels of blood glucose. 175 00:16:08 --> 00:16:14 As you probably know, 176 00:16:14 --> 00:16:17 your body stores energy different ways. One way is in a polymer of 177 00:16:17 --> 00:16:20 glucose called glycogen, which is stored in your liver. 178 00:16:20 --> 00:16:24 Another way is through the storage of fat. 179 00:16:24 --> 00:16:30 For this response we want to liberate the energy that's stored in 180 00:16:30 --> 00:16:36 the form of glycogen and turn it into glucose which is much more 181 00:16:36 --> 00:16:42 consumable. It can be used directly by your muscles to make ATP which 182 00:16:42 --> 00:16:48 drives various reactions like contractions and so on. 183 00:16:48 --> 00:16:54 So the analogy here is money in your bank account versus 184 00:16:54 --> 00:17:00 money in your wallet. When you're in a situation like that, 185 00:17:00 --> 00:17:04 you want to transfer money from your bank account to your wallet. 186 00:17:04 --> 00:17:08 That's the direction we're heading in this example. 187 00:17:08 --> 00:17:12 In others situations, where you've just had a huge meal 188 00:17:12 --> 00:17:16 and you're not running from a bear, you go the other way. You take 189 00:17:16 --> 00:17:20 glucose and you turn it into glycogen, which I said is a polymer 190 00:17:20 --> 00:17:24 of glucose. Now, this balance between glycogen and 191 00:17:24 --> 00:17:28 glucose is controlled by enzymes which are sort of in the center of 192 00:17:28 --> 00:17:33 today's example. In this direction the enzyme is 193 00:17:33 --> 00:17:39 glycogen phosphorylase. Glycogen phosphorylase is the last 194 00:17:39 --> 00:17:45 enzyme that breaks down glycogen into a form of glucose that can be 195 00:17:45 --> 00:17:51 then further metabolized. Glycogen phosphorylase. And the 196 00:17:51 --> 00:17:57 other, in the other direction, is an enzyme called glycogen 197 00:17:57 --> 00:18:02 synthase. OK? So the goal of this reaction, 198 00:18:02 --> 00:18:06 or set of signals, will be to stimulate this process and actually 199 00:18:06 --> 00:18:10 inhibit this process. And you'll see how that's done in a 200 00:18:10 --> 00:18:14 second. This is epinephrine. Again, it's a small chemical 201 00:18:14 --> 00:18:19 produced by your adrenal glands. As I said earlier, it's released 202 00:18:19 --> 00:18:23 during the flight or fight response. It binds to a class of receptors 203 00:18:23 --> 00:18:27 which I'll take you through. They're called beta-adrenergic 204 00:18:27 --> 00:18:31 receptors because they bind to a factor 205 00:18:31 --> 00:18:35 produced in the adrenal glands, beta-adrenergic receptors. These 206 00:18:35 --> 00:18:39 are receptors, as you'll see, that have a complex 207 00:18:39 --> 00:18:43 tortuous root through the membrane. They go through the membrane seven 208 00:18:43 --> 00:18:46 times. They're actually sometimes referred to as serpentine receptors 209 00:18:46 --> 00:18:50 for that reason. And they bind to and activate GTP 210 00:18:50 --> 00:18:54 binding proteins, G proteins, as I mentioned down here. 211 00:18:54 --> 00:18:58 And this is a particular form of G protein called trimeric G proteins. 212 00:18:58 --> 00:19:01 And you'll see why in a second. As I mentioned, 213 00:19:01 --> 00:19:04 epinephrine binds to cells in different parts of the body 214 00:19:04 --> 00:19:07 resulting in various physiological effects. The ones I listed here and 215 00:19:07 --> 00:19:11 another one which I didn't list here which I find paradoxical. 216 00:19:11 --> 00:19:14 Actually, I know what, I know what the evolutionary advantage to all of 217 00:19:14 --> 00:19:17 these things is, but I don't know what the 218 00:19:17 --> 00:19:20 evolutionary advantage is to the relaxation of the smooth muscles 219 00:19:20 --> 00:19:23 around the colon during the fight or flight response. 220 00:19:23 --> 00:19:26 Some of you may have felt this before when you're extremely fearful. 221 00:19:26 --> 00:19:30 I'm not sure why it is our bodies do that. 222 00:19:30 --> 00:19:35 But anyway. OK. So how does this work? 223 00:19:35 --> 00:19:47 So we're going to consider just the 224 00:19:47 --> 00:19:52 mobilization of glucose. And the site of action is the liver. 225 00:19:52 --> 00:19:57 That's where glycogen is stored. And on the cells in the liver, the 226 00:19:57 --> 00:20:02 hepatocytes, on the plasma membrane of those cells, 227 00:20:02 --> 00:20:07 there are these receptors, these serpentine receptors. 228 00:20:07 --> 00:20:11 They look like that. They look like snakes, 229 00:20:11 --> 00:20:16 as I mentioned. Beta-adrenergic receptors. There are also 230 00:20:16 --> 00:20:20 alpha-adrenergic receptors, but we're going to focus on 231 00:20:20 --> 00:20:25 beta-adrenergic receptors. And these bind to epinephrine 232 00:20:25 --> 00:20:30 directly. So here's our molecule of epinephrine. 233 00:20:30 --> 00:20:37 It physically binds to a portion of 234 00:20:37 --> 00:20:43 the polypeptide chain that's sticking out from the cell's surface. 235 00:20:43 --> 00:20:48 Now, bound on the inside to this receptor is this trimeric G protein. 236 00:20:48 --> 00:20:54 It's called trimeric because there are three subunits. 237 00:20:54 --> 00:21:00 There's an alpha subunit, a beta subunit and a gamma subunit. 238 00:21:00 --> 00:21:06 And in this configuration, prior to the binding of epinephrine, 239 00:21:06 --> 00:21:12 the alpha subunit is bound to GDP. And, as I mentioned before, 240 00:21:12 --> 00:21:19 that's usually associated with inactivity. So the alpha subunit is 241 00:21:19 --> 00:21:25 inactive. The binding of epinephrine to the surface has two 242 00:21:25 --> 00:21:31 effects. The first is an exchange wherein the alpha subunit 243 00:21:31 --> 00:21:37 now binds GTP. And the GDP that was present there 244 00:21:37 --> 00:21:42 comes off. The other effect is that the G protein, 245 00:21:42 --> 00:21:47 the alpha subunit releases from the beta and gamma subunits and they 246 00:21:47 --> 00:21:53 float away. In this, now GTP-bound configuration, 247 00:21:53 --> 00:21:58 the alpha subunit is now active. And it can move from this position 248 00:21:58 --> 00:22:03 to another position within the membrane and bind to 249 00:22:03 --> 00:22:09 another protein. In this case an enzyme called 250 00:22:09 --> 00:22:16 adenylate cyclase. The GTP bound version of the alpha 251 00:22:16 --> 00:22:23 subunit associates with adenylate cyclase, thereby converting it to an 252 00:22:23 --> 00:22:31 active form. When bound to this it becomes active. 253 00:22:31 --> 00:22:37 And the consequence of that is that the adenylate cyclase can now 254 00:22:37 --> 00:22:43 convert ATP to the second messenger cyclic AMP. OK? 255 00:22:43 --> 00:22:49 And this process that we've gone through from binding of a single 256 00:22:49 --> 00:22:56 molecule to the production of a lot of a cyclic AMP is an example. 257 00:22:56 --> 00:23:18 OK? We've made a lot of second 258 00:23:18 --> 00:23:22 messenger from the binding of a single molecule because we've turned 259 00:23:22 --> 00:23:25 on a lot of these, which can go on and turn on a lot of 260 00:23:25 --> 00:23:29 these, which can go on and make a lot of this. 261 00:23:29 --> 00:23:40 Now, there's another step in signal 262 00:23:40 --> 00:23:48 amplification here in this pathway. There's an enzyme known as protein 263 00:23:48 --> 00:23:57 kinase A which becomes activated in the presence of cyclic AMP. 264 00:23:57 --> 00:24:05 So protein kinase A in the presence of cyclic AMP goes from an inactive 265 00:24:05 --> 00:24:13 state to an active state. And when it becomes active it 266 00:24:13 --> 00:24:21 converts another enzyme, glycogen phosphorylase, which I 267 00:24:21 --> 00:24:29 mentioned to you a moment ago, from an inactive state to an active 268 00:24:29 --> 00:24:43 state. 269 00:24:43 --> 00:24:46 OK? So this is a phosphorylation reaction, similar to the one that I 270 00:24:46 --> 00:24:50 drew up here. PKA phosphorylates glycogen phosphorylase, 271 00:24:50 --> 00:24:54 making it go from an inactive to an active state. Another step of 272 00:24:54 --> 00:24:58 signal amplification. We can make some of this active 273 00:24:58 --> 00:25:03 which makes a lot of this active. OK? And this is the enzyme we 274 00:25:03 --> 00:25:09 needed to turn on in order to break down glycogen to glucose. 275 00:25:09 --> 00:25:16 Now, there's another kind of cool thing that happens in the control of 276 00:25:16 --> 00:25:22 the reverse reaction, because it turns out that the enzyme 277 00:25:22 --> 00:25:28 responsible for making glycogen, glycogen synthase is inactivated in 278 00:25:28 --> 00:25:35 the presence of cyclic AMP. So it goes from an active state in 279 00:25:35 --> 00:25:41 the presence of cyclic AMP to an inactive state. 280 00:25:41 --> 00:25:49 So at once we turn that off, 281 00:25:49 --> 00:25:54 turn this on, and we can generate a lot of glucose. 282 00:25:54 --> 00:25:59 OK. So this is the reaction. We focused exclusively on the 283 00:25:59 --> 00:26:03 mobilization of glucose here. Just to remind you, 284 00:26:03 --> 00:26:06 this same pathway controls heart rate, breathing rate, 285 00:26:06 --> 00:26:10 blood flow. And, actually, some of you might have heard of 286 00:26:10 --> 00:26:13 beta-blockers. And beta-blockers are used to 287 00:26:13 --> 00:26:16 control this exact reaction. Beta-blockers bind to 288 00:26:16 --> 00:26:19 beta-adrenergic receptors so that your body won't respond to 289 00:26:19 --> 00:26:23 adrenaline or epinephrine when you don't want it to. 290 00:26:23 --> 00:26:28 So people who get really nervous talking in front of audiences or 291 00:26:28 --> 00:26:33 taking exams in 7. 1 will sometimes take beta-blockers 292 00:26:33 --> 00:26:38 so that they don't suffer, you know, fast heart rate or this 293 00:26:38 --> 00:26:44 feeling of anxiety and so on. OK. So we've turned on this signal 294 00:26:44 --> 00:26:49 in response to binding of epinephrine to the cell's surface. 295 00:26:49 --> 00:26:54 We've started all these happening. How do we turn it off? How do we 296 00:26:54 --> 00:27:00 deal with signal termination? How do we turn the signal off? 297 00:27:00 --> 00:27:05 Anybody have any ideas? Well, one might be not so obvious. 298 00:27:05 --> 00:27:16 And that is -- 299 00:27:16 --> 00:27:19 -- a process which I introduced to you last time called pathway 300 00:27:19 --> 00:27:23 modulation in which the activation of a second pathway can actually 301 00:27:23 --> 00:27:27 inhibit the activation of the primary pathway. 302 00:27:27 --> 00:27:32 So, for example, I've been telling you about the 303 00:27:32 --> 00:27:37 beta-adrenergic receptors, which through the binding of this 304 00:27:37 --> 00:27:42 kicks off a series of events which results in the activation of this 305 00:27:42 --> 00:27:48 pathway. There are many examples in biology where the binding of a 306 00:27:48 --> 00:27:53 second ligand to a distinct receptor results in the propagation of a 307 00:27:53 --> 00:27:59 signal which actually inhibits the first. 308 00:27:59 --> 00:28:02 So, for example, I mentioned here that there's a 309 00:28:02 --> 00:28:06 kinase that's activated, protein kinase A. This pathway 310 00:28:06 --> 00:28:10 might activate the relevant phosphatase, thereby turning this 311 00:28:10 --> 00:28:13 signal down. So modulation of the pathway, very important in biology. 312 00:28:13 --> 00:28:17 We're only really beginning to appreciate how important it is the 313 00:28:17 --> 00:28:21 more we learn. Another obvious example is ligand 314 00:28:21 --> 00:28:25 diffusion. You make a certain amount of ligand from 315 00:28:25 --> 00:28:30 your adrenal glands. And it just diffuses eventually or 316 00:28:30 --> 00:28:36 breaks down, thereby turning the signal off. There's also a 317 00:28:36 --> 00:28:43 phenomenon known as receptor internalization. 318 00:28:43 --> 00:28:51 Very often when a receptor binds to 319 00:28:51 --> 00:28:55 its ligand, it doesn't just stay out there on the surface but actually 320 00:28:55 --> 00:28:59 gets internalized inside the cell. So I'll draw a different class of 321 00:28:59 --> 00:29:03 receptors here. Here's the receptor. 322 00:29:03 --> 00:29:07 Here it's bound to its ligand. I told you last time that proteins 323 00:29:07 --> 00:29:11 can get to the cell's surface through a complex sorting pathway 324 00:29:11 --> 00:29:16 that involves actually vesicles encapsulating the proteins and then 325 00:29:16 --> 00:29:20 bringing them to the cell surface. Well, the reverse can happen as 326 00:29:20 --> 00:29:25 well where the proteins can be removed from the cell's surface into 327 00:29:25 --> 00:29:29 vesicles. Removing them from the cell's surface then takes them away 328 00:29:29 --> 00:29:33 from the possibility of being activated, and thereby turning 329 00:29:33 --> 00:29:38 down the signal. Another possibility would be to 330 00:29:38 --> 00:29:43 degrade the second messenger. OK? Cyclic AMP is an example. 331 00:29:43 --> 00:29:47 You need to make it in order to stimulate these various downstream 332 00:29:47 --> 00:29:52 events, so get rid of it. And, in fact, there's a clear 333 00:29:52 --> 00:29:57 pathway for that process. As I told you, ATP can be converted 334 00:29:57 --> 00:30:02 through the actions of adenylate cyclase to make cyclic AMP. 335 00:30:02 --> 00:30:10 But likewise there's an enzyme, actually, a whole class of enzymes 336 00:30:10 --> 00:30:18 known as phosphodiesterases which converts cyclic AMP to AMP, 337 00:30:18 --> 00:30:26 thereby inactivating the signal. These enzymes are actually very 338 00:30:26 --> 00:30:32 important in pharmacology. There are lots of inhibitors of 339 00:30:32 --> 00:30:36 phosphodiesterases which stimulate the production or the maintenance of 340 00:30:36 --> 00:30:40 high levels of cyclic AMP and other cyclic nucleotides. 341 00:30:40 --> 00:30:44 So caffeine for example, one of the reasons you get a buzz 342 00:30:44 --> 00:30:48 from caffeine is that it blocks a class of these enzymes leading to 343 00:30:48 --> 00:30:52 more cyclic AMP which gives you that kind of nervous energy. 344 00:30:52 --> 00:30:56 OK? Another related process involving another cyclic nucleotide, 345 00:30:56 --> 00:31:01 this time cyclic GTP. This is broken down by a different 346 00:31:01 --> 00:31:07 phosphodiesterase. In Viagra and like chemicals are 347 00:31:07 --> 00:31:14 inhibitors of this class of phosphodiesterases, 348 00:31:14 --> 00:31:20 thereby leading to an over stimulation of that pathway. 349 00:31:20 --> 00:31:27 And, finally, you can reverse the structural changes that I mentioned 350 00:31:27 --> 00:31:33 previously. So I said earlier that enzymes can be activated 351 00:31:33 --> 00:31:42 through kinases. And they can be inactivated by 352 00:31:42 --> 00:31:52 phosphatases. I'm going to have to move to a different board here. 353 00:31:52 --> 00:32:02 And G proteins which get activated in the beginning of these processes, 354 00:32:02 --> 00:32:12 from a GDP bound form, through the action of exchange factors to the 355 00:32:12 --> 00:32:22 GTP bound form, can be inactivated by GTP hydrolysis. 356 00:32:22 --> 00:32:26 And this is often through proteins called GAPs, which are called GTPase 357 00:32:26 --> 00:32:31 activating proteins. They activate the GTP hydrolysis 358 00:32:31 --> 00:32:36 activity of the G protein, thereby returning the G protein to 359 00:32:36 --> 00:32:40 its inactive state. OK. So that's the beta-adrenergic 360 00:32:40 --> 00:32:45 signaling pathway. It's a classic. You should know it 361 00:32:45 --> 00:32:50 in principle and in some detail. It's covered extensively in your 362 00:32:50 --> 00:32:55 book. Here's a figure from your book. 363 00:32:55 --> 00:32:58 I won't go through the details of this because we just did them on the 364 00:32:58 --> 00:33:01 board, but just so you know it's in there. Here's the production of the 365 00:33:01 --> 00:33:04 second messenger, cyclic AMP. And then it triggers 366 00:33:04 --> 00:33:07 this kinase cascade that I drew up for you before, 367 00:33:07 --> 00:33:10 activation of PKA, activation of glycogen phosphorylase, 368 00:33:10 --> 00:33:13 and the production ultimately of glucose. And here's the 369 00:33:13 --> 00:33:16 second messenger. I'm pleased to see that I put this 370 00:33:16 --> 00:33:19 in the book, in my slide set last year because the book had an 371 00:33:19 --> 00:33:23 incorrect structure for cyclic AMP in the previous edition. 372 00:33:23 --> 00:33:26 I'm pleased to see they know have corrected the structure. 373 00:33:26 --> 00:33:30 This is the actual proper structure of cyclic AMP. 374 00:33:30 --> 00:33:34 OK. So now want to change signaling pathways and consider not what you 375 00:33:34 --> 00:33:39 do when you first encounter the bear, but what do you do, 376 00:33:39 --> 00:33:44 or what does your body do if you fail to successfully run from the 377 00:33:44 --> 00:33:49 bear? So it's funny what you can find on the Web, 378 00:33:49 --> 00:33:53 boy. You can find anything you want on the Web. In fact, 379 00:33:53 --> 00:33:58 when I was looking up, I was pretty sure that Viagra 380 00:33:58 --> 00:34:02 blocked phosphodiesterases. I was pretty sure, 381 00:34:02 --> 00:34:06 but I wasn't certain so I did a Web search. This is true. 382 00:34:06 --> 00:34:10 I did a Web search. And, God, I cannot believe what I came 383 00:34:10 --> 00:34:14 across. It's unbelievable. But you can find some pretty nasty 384 00:34:14 --> 00:34:17 bear wounds on the Web as well, and here's one of them. So this guy 385 00:34:17 --> 00:34:21 didn't run fast enough. So what's going to happen? 386 00:34:21 --> 00:34:25 What is his body going to do following an injury such as that? 387 00:34:25 --> 00:34:29 Well, obviously he needs to heal the wound. 388 00:34:29 --> 00:34:33 Your body is remarkable in its ability to detect damage and fix it. 389 00:34:33 --> 00:34:37 So we're going to heal the wound. And this is an example of one of 390 00:34:37 --> 00:34:42 these long-term effects of a signaling pathway. 391 00:34:42 --> 00:34:46 Whereas, this was short-term, everything was happening in the 392 00:34:46 --> 00:34:50 cytoplasm, generation of glucose to be secreted. This is a long-term 393 00:34:50 --> 00:34:55 effect. The effect has to take place ultimately in the nucleus 394 00:34:55 --> 00:34:59 because we need to turn on gene expression to convince cells to 395 00:34:59 --> 00:35:04 divide and heal the wound. This may be a troubling picture. 396 00:35:04 --> 00:35:10 Maybe I should go back to the bear. There. So if we imagine the skin, 397 00:35:10 --> 00:35:16 which is a tight complex of cells forming a protective layer against 398 00:35:16 --> 00:35:21 the elements and so on keeping stuff out that should be out, 399 00:35:21 --> 00:35:27 in that should be in. In a wound you breach that structure, 400 00:35:27 --> 00:35:33 you get a scratch. And one of the things that happens, 401 00:35:33 --> 00:35:40 as you bleed into that wound, is that small packets, 402 00:35:40 --> 00:35:48 former bits of cells called platelets bind to the edges of that 403 00:35:48 --> 00:35:55 wound. And the platelets then release a factor imaginatively 404 00:35:55 --> 00:36:03 called platelet-derived growth factor. 405 00:36:03 --> 00:36:09 And platelet-derived growth factor, otherwise known as PDGF stimulates 406 00:36:09 --> 00:36:15 other cells to divide. And specifically it stimulates 407 00:36:15 --> 00:36:22 cells called fibroblasts which sit underneath these cells normally as 408 00:36:22 --> 00:36:28 part of the connective tissue underneath the epithelial cells. 409 00:36:28 --> 00:36:35 In the presence of PDGF, the fibroblasts proliferate. 410 00:36:35 --> 00:36:39 And this results in the formation of a scar. OK? And what we want to 411 00:36:39 --> 00:36:44 understand now is how is it that PDGF stimulates fibroblasts 412 00:36:44 --> 00:37:21 to divide? 413 00:37:21 --> 00:37:29 So we can model this process in cell culture. We can extract fibroblasts 414 00:37:29 --> 00:37:37 from your skin and put them in a tissue culture dish, 415 00:37:37 --> 00:37:45 let's say one times ten to the fifth cells. 416 00:37:45 --> 00:37:51 So this is cell number on this axis and this is days in culture on this 417 00:37:51 --> 00:37:57 axis, one, two, three, four days. 418 00:37:57 --> 00:38:03 If we plate 100,000 fibroblasts in a tissue culture dish, 419 00:38:03 --> 00:38:09 in the absence of growth factors they won't divide. 420 00:38:09 --> 00:38:14 They'll just sit there. But if we add PDGF or other growth 421 00:38:14 --> 00:38:19 factors, the cells will divide, and therefore we'll see an increase 422 00:38:19 --> 00:38:24 in cell number. So, again, the question is how does 423 00:38:24 --> 00:38:29 that happen? What is the mechanism that allows that to happen? 424 00:38:29 --> 00:38:43 Oops. What did I do? 425 00:38:43 --> 00:38:48 Well, here we're talking about another class of receptors. 426 00:38:48 --> 00:38:54 These are called growth factor receptors. And these are different 427 00:38:54 --> 00:38:59 from seven transmembrane receptors in that they have, 428 00:38:59 --> 00:39:05 on their cytoplasmic side, enzymatic activity, specifically 429 00:39:05 --> 00:39:10 kinase domains. These receptors are also enzymes. 430 00:39:10 --> 00:39:16 And their enzymatic activity is to add phosphates to other proteins. 431 00:39:16 --> 00:39:21 In this configuration, however, they're inactive. 432 00:39:21 --> 00:39:27 What the growth factor does is causes those receptors to dimerize 433 00:39:27 --> 00:39:33 with one another. To become in close proximity to one 434 00:39:33 --> 00:39:39 another. So the growth factor serves to link them. 435 00:39:39 --> 00:39:46 Here's our growth factor. And in this now increased proximity, 436 00:39:46 --> 00:39:53 these kinase domains become active. And the consequence of that is we 437 00:39:53 --> 00:40:00 get phosphorylation of the neighboring protein. 438 00:40:00 --> 00:40:11 We call this transphosphorylation. 439 00:40:11 --> 00:40:14 So ligand induced activation, transphosphorylation. And now what 440 00:40:14 --> 00:40:18 we've done, on the inside of the cell, is to create a structure. 441 00:40:18 --> 00:40:22 And it's the formation of that structure that then allows the rest 442 00:40:22 --> 00:40:51 of the process to work. 443 00:40:51 --> 00:40:56 These phosphorylated residues act as the binding sites for other proteins. 444 00:40:56 --> 00:41:01 The first one is known as an adaptor protein. 445 00:41:01 --> 00:41:10 An adaptor protein which binds to a 446 00:41:10 --> 00:41:16 class of proteins to that I referred to previously, 447 00:41:16 --> 00:41:22 a guanine nucleotide exchange factor. And that then allows the exchange 448 00:41:22 --> 00:41:28 factor to stimulate the exchange of a small GTP binding protein, 449 00:41:28 --> 00:41:34 which is generally bound to GDP and inactive, to stimulate the exchange 450 00:41:34 --> 00:41:40 of GTP for GDP. This small GTP binding protein is a 451 00:41:40 --> 00:41:45 protein called RAS, a very important signaling molecule. 452 00:41:45 --> 00:41:50 And, actually, a very important cancer molecule as well. 453 00:41:50 --> 00:41:55 Mutationally activated in a high percentage of cancers. 454 00:41:55 --> 00:42:00 When you now have RAS in its GTP bound state, it can activate 455 00:42:00 --> 00:42:05 a kinase cascade. Which I'll go through with you on 456 00:42:05 --> 00:42:11 slides in a moment. It first activates a protein called 457 00:42:11 --> 00:42:16 RAF which then activates a protein called MEK which then activates a 458 00:42:16 --> 00:42:21 protein called ERK. ERK is a map kinase. 459 00:42:21 --> 00:42:27 It's a kinase itself. Its enzymatic activity is to 460 00:42:27 --> 00:42:33 phosphorylate other proteins. MEK phosphorylates ERK so it's 461 00:42:33 --> 00:42:40 called a map kinase kinase. And RAF is a kinase itself which 462 00:42:40 --> 00:42:47 phosphorylates this kinase so it's referred to as a map kinase kinase 463 00:42:47 --> 00:42:54 kinase. I'm not making it up. OK? So it stimulates a kinase 464 00:42:54 --> 00:43:02 cascade, similar to what I introduced you to before. 465 00:43:02 --> 00:43:06 And then, as you'll see, ERK, which is a cytoplasmic protein, 466 00:43:06 --> 00:43:11 moves from the cytoplasm into the nucleus. I'll show this to you on 467 00:43:11 --> 00:43:16 slides, and it's in your book as well. And in the nucleus this 468 00:43:16 --> 00:43:21 kinase phosphorylates transcription factors which then bind to target 469 00:43:21 --> 00:43:26 genes turning them on stimulating the cell to divide. 470 00:43:26 --> 00:43:31 I would have drawn that for you but we're running out of time. 471 00:43:31 --> 00:43:35 So I will show it to you instead on the overheads. 472 00:43:35 --> 00:43:39 There's our wound. So here's the pathway. 473 00:43:39 --> 00:43:43 It's just straight out of your book. Here are the growth factor 474 00:43:43 --> 00:43:47 receptors, again, not dimerized, not active. 475 00:43:47 --> 00:43:51 The growth factor causes the two proteins to come together in close 476 00:43:51 --> 00:43:55 apposition. This causes the kinase activities to get stimulated 477 00:43:55 --> 00:44:00 resulting in the phosphorylation of the cytoplasmic tails. 478 00:44:00 --> 00:44:04 The adaptor protein then can bind to the phosphorylated tail. 479 00:44:04 --> 00:44:09 This then stimulates through an exchange factor not shown here, 480 00:44:09 --> 00:44:14 the exchange of GDP for GTP, making an active RAS molecule. 481 00:44:14 --> 00:44:19 This then can activate RAF making it now an active kinase which 482 00:44:19 --> 00:44:24 activates MEK making it an active kinase which activates ERK called 483 00:44:24 --> 00:44:28 map K here for map kinase. This now goes into the nucleus of 484 00:44:28 --> 00:44:32 the cell from the cytoplasm, and in the cytoplasm it can 485 00:44:32 --> 00:44:36 encounter transcription factors. It then phosphorylates those 486 00:44:36 --> 00:44:40 transcription factors which bind to their target genes turning on gene 487 00:44:40 --> 00:44:44 expression and leading to a change in the cellular phenotype. 488 00:44:44 --> 00:44:48 And the change in the cellular phenotype that we're talking about 489 00:44:48 --> 00:44:52 here is cell division, the details of which we'll cover 490 00:44:52 --> 00:44:56 next time. So this is sort of a static picture. 491 00:44:56 --> 00:45:00 But to give you a more dynamic picture of what's happening here, 492 00:45:00 --> 00:45:05 I borrowed from the Biocreations Website an animation, 493 00:45:05 --> 00:45:10 which I find very helpful and, actually, is kind of funny as well. 494 00:45:10 --> 00:45:15 So we're going to start this process much the same way that we've 495 00:45:15 --> 00:45:20 just talked about using the terms that I used on the board. 496 00:45:20 --> 00:45:25 Oop. We need sound. Let's do that again so you can hear it 497 00:45:25 --> 00:45:29 in all its glory. OK. So the first thing is the receptors 498 00:45:29 --> 00:45:33 get dimerized by the growth factor leading to the phosphorylation of 499 00:45:33 --> 00:45:37 these cytoplasmic tails. And then this protein, which is the 500 00:45:37 --> 00:45:41 adaptor, it's called grab-2 grabs onto one of those phosphorylation 501 00:45:41 --> 00:45:45 sites. It has attached to it an exchange factor called SAS here. 502 00:45:45 --> 00:45:49 OK? So that's the first thing that happens. You recruit this signaling 503 00:45:49 --> 00:45:53 complex to the membrane. Once you have it there it's now in 504 00:45:53 --> 00:45:57 close proximity to RAS which is, if you remember, a membrane 505 00:45:57 --> 00:46:01 associated protein. It's now in its inactive stage, 506 00:46:01 --> 00:46:06 its GDP bound state. Oops. Sorry. We'll just have to 507 00:46:06 --> 00:46:13 watch that again. Oh, no. Wait. Wait. 508 00:46:13 --> 00:46:19 Wait. Go back. Forget you saw that. That's the end. 509 00:46:19 --> 00:46:26 You never want to see the ending. Oh, wait a minute. OK. Here we go. 510 00:46:26 --> 00:46:32 OK. So next we're going to activate RAS. 511 00:46:32 --> 00:46:36 It gets a little kick in the butt. GDP comes off. 512 00:46:36 --> 00:46:41 GTP comes on. Now RAS is active. It's now capable of interacting 513 00:46:41 --> 00:46:46 productively with RAF, the first of these kinases, 514 00:46:46 --> 00:46:50 the map kinase kinase kinase. And it's going to cause it to 515 00:46:50 --> 00:46:55 become active. Once it's active it's now capable 516 00:46:55 --> 00:47:00 of activating the next guy in line, which is MEK. 517 00:47:00 --> 00:47:03 Now MEK is active, and it's going to phosphorylate ERK, 518 00:47:03 --> 00:47:06 and now ERK is phosphorylated and active. Now, there have been a 519 00:47:06 --> 00:47:09 couple of other steps, which you can look at, at home, 520 00:47:09 --> 00:47:12 but the RAS went from a GTP bound stage to a GDP bound state. 521 00:47:12 --> 00:47:15 I told you that you can turn these signals off up here. 522 00:47:15 --> 00:47:18 That happens in this pathway. Another GAP comes along and turns 523 00:47:18 --> 00:47:21 RAS off. And here's a protein phosphatase, which is going to clip 524 00:47:21 --> 00:47:24 off the phosphate, thereby turning MEK off. 525 00:47:24 --> 00:47:28 So each of these steps is reversible. 526 00:47:28 --> 00:47:35 But, importantly, we're down here at this level where 527 00:47:35 --> 00:47:42 ERK has been phosphorylated. And now it's capable, dammit. 528 00:47:42 --> 00:47:49 All right. We'll have to watch it again. Wait a minute. 529 00:47:49 --> 00:47:56 Come on. OK. Here we go. I know. It's like a video game. 530 00:47:56 --> 00:48:02 OK. So ERK is active. 531 00:48:02 --> 00:48:06 And now it's going to go in the nucleus. Phosphorylate Jun, 532 00:48:06 --> 00:48:10 one of these transcription factors. And upon doing so the transcription 533 00:48:10 --> 00:48:14 factors dimerize, bind to the promoter of a target 534 00:48:14 --> 00:48:18 gene. They recruit RNA polymerase. And this leads to the production of 535 00:48:18 --> 00:48:22 new gene expression. mRNAs that go into the cytoplasm 536 00:48:22 --> 00:48:26 get translated into proteins that convince the cell now it's 537 00:48:26 --> 00:48:29 time to divide. OK? So I encourage you to look at this 538 00:48:29 --> 00:48:33 to get the details. Now, if you just wait one more 539 00:48:33 --> 00:48:37 second. In the last 30 seconds I want to point out the fact that 540 00:48:37 --> 00:48:41 these pathways, while we're teaching to you as 541 00:48:41 --> 00:48:44 simple linear pathways, are actually highly complex. 542 00:48:44 --> 00:48:48 Pay attention to this clock here. It's 11:54. Highly complex, highly 543 00:48:48 --> 00:48:52 interactive, not linear, and therefore we frequently 544 00:48:52 --> 00:48:56 analogize what's happening inside a living cell to an integrated 545 00:48:56 --> 00:49:00 circuit. And increasingly these days in 546 00:49:00 --> 00:49:04 biology, we're thinking about these things in analogy with computers, 547 00:49:04 --> 00:49:09 and actually relying extensively on computers to help us understand how 548 00:49:09 --> 00:49:13 that complexity actually works using methods from chemistry and physics 549 00:49:13 --> 00:49:18 and mathematics and other disciplines in a new and emerging 550 00:49:18 --> 00:49:22 area called systems biology, which you might be interested in. 551 00:49:22 --> 00:49:27 And the new Biological Engineering major will have a strong focus 552 00:49:27 --> 00:49:30 in this area. OK.