1 00:00:15,680 --> 00:00:18,110 PROFESSOR: OK, here we go. 2 00:00:18,110 --> 00:00:19,100 Couple of things. 3 00:00:19,100 --> 00:00:22,070 Sorry, I forgot to bring candy, but it'll be on sale next week. 4 00:00:22,070 --> 00:00:24,070 So we can probably bring it next week. 5 00:00:24,070 --> 00:00:27,380 But walking over, I thought, boy, those of you who are here 6 00:00:27,380 --> 00:00:28,470 deserve some candy. 7 00:00:28,470 --> 00:00:32,570 But I've just tried to sprinkle in a few interesting slides 8 00:00:32,570 --> 00:00:34,230 for your benefit. 9 00:00:34,230 --> 00:00:36,380 I saw this on the MIT news of the day, 10 00:00:36,380 --> 00:00:38,420 and I thought that was really cool. 11 00:00:38,420 --> 00:00:41,960 Who would have thought to turn the giant dome into a Halloween 12 00:00:41,960 --> 00:00:42,890 pumpkin? 13 00:00:42,890 --> 00:00:44,690 And I was also jealous this morning 14 00:00:44,690 --> 00:00:48,380 when my husband got ready to go work in the emergency room 15 00:00:48,380 --> 00:00:50,660 and he put on his Star Trek outfit. 16 00:00:50,660 --> 00:00:53,780 So I was like, oh, I didn't even have 17 00:00:53,780 --> 00:00:55,850 a-- because I don't usually get to actually 18 00:00:55,850 --> 00:00:58,070 have a class on the day of Halloween. 19 00:00:58,070 --> 00:01:00,230 So he headed off. 20 00:01:00,230 --> 00:01:01,850 I think people are unfortunately going 21 00:01:01,850 --> 00:01:04,849 to expect him to be able to really fix things very readily 22 00:01:04,849 --> 00:01:07,790 in the emergency room today because he's 23 00:01:07,790 --> 00:01:10,400 going to have all those extra powers that he doesn't usually 24 00:01:10,400 --> 00:01:11,630 have. 25 00:01:11,630 --> 00:01:14,720 But anyway, so actually it's kind 26 00:01:14,720 --> 00:01:18,320 of a good day for a lecture, Halloween, 27 00:01:18,320 --> 00:01:22,400 because we're going to talk about the fight or flight 28 00:01:22,400 --> 00:01:25,400 response, which is a great paradigm 29 00:01:25,400 --> 00:01:26,590 for cellular signaling. 30 00:01:26,590 --> 00:01:29,240 So you're going to see how signaling really 31 00:01:29,240 --> 00:01:30,980 works in action. 32 00:01:30,980 --> 00:01:33,470 Because what one has to think about 33 00:01:33,470 --> 00:01:35,690 with respect to cellular signaling 34 00:01:35,690 --> 00:01:40,920 is that it's dynamic and transient. 35 00:01:43,950 --> 00:01:46,410 And when we look at the molecular details 36 00:01:46,410 --> 00:01:50,910 of the switches that enable dynamics and transient behavior 37 00:01:50,910 --> 00:01:54,060 in cells, you're going to see how perfectly adapted 38 00:01:54,060 --> 00:01:57,030 they are for these types of responses that 39 00:01:57,030 --> 00:02:00,150 have to be carried out in cells or in organs 40 00:02:00,150 --> 00:02:04,080 in order to respond to a particular signal rapidly 41 00:02:04,080 --> 00:02:06,750 and with a definitive time frame and then 42 00:02:06,750 --> 00:02:10,750 have that signal then stop once the time frame has passed. 43 00:02:10,750 --> 00:02:13,760 So I really want to sort of stress to you 44 00:02:13,760 --> 00:02:19,050 the characteristics of signaling that can emerge just 45 00:02:19,050 --> 00:02:24,390 by knowing about two particular cellular switches, 46 00:02:24,390 --> 00:02:27,300 knowing the molecular details of those switches, 47 00:02:27,300 --> 00:02:30,840 to really understand, then, when we look at them in action 48 00:02:30,840 --> 00:02:33,600 in a couple of cellular signaling pathways, 49 00:02:33,600 --> 00:02:36,570 then we'll see how adapted those signals are. 50 00:02:36,570 --> 00:02:38,880 And the great thing about biology 51 00:02:38,880 --> 00:02:42,870 is that once you learn a few very specific things, 52 00:02:42,870 --> 00:02:45,900 then those often get reused in nature. 53 00:02:45,900 --> 00:02:48,870 So the cellular signals that I'm going to describe to you 54 00:02:48,870 --> 00:02:51,810 are used again and again in different formats 55 00:02:51,810 --> 00:02:54,390 to create different signaling pathways. 56 00:02:54,390 --> 00:02:56,520 So it's not at every pathway. 57 00:02:56,520 --> 00:02:59,610 And every cell in the body has different nuances. 58 00:02:59,610 --> 00:03:04,020 It has general paradigms that we can learn about and understand, 59 00:03:04,020 --> 00:03:04,680 all right? 60 00:03:04,680 --> 00:03:10,320 So the key feature is then to think about the molecular basis 61 00:03:10,320 --> 00:03:11,590 of these switches. 62 00:03:11,590 --> 00:03:14,940 So last time, I was talking to you about cellular signaling. 63 00:03:14,940 --> 00:03:25,760 And remember, there's always a signal, a response, 64 00:03:25,760 --> 00:03:26,350 and an output. 65 00:03:29,180 --> 00:03:30,440 So something happens. 66 00:03:30,440 --> 00:03:31,190 It's a signal. 67 00:03:31,190 --> 00:03:35,570 It's usually a molecule of some kind outside the cell 68 00:03:35,570 --> 00:03:38,150 or able to diffuse into the cell. 69 00:03:38,150 --> 00:03:41,220 As a function of that signal, there's a response. 70 00:03:41,220 --> 00:03:42,440 So this is molecular. 71 00:03:46,640 --> 00:03:49,390 The response obviously is biochemical. 72 00:03:53,430 --> 00:03:55,650 And the output is biological. 73 00:03:58,840 --> 00:04:01,050 So I want you to sort of think about these 74 00:04:01,050 --> 00:04:03,090 as we look at pathways. 75 00:04:03,090 --> 00:04:05,220 What's really my output at the end 76 00:04:05,220 --> 00:04:07,230 of a particular signaling pathway? 77 00:04:07,230 --> 00:04:08,430 What was my input? 78 00:04:08,430 --> 00:04:10,350 How did it get to the cell? 79 00:04:10,350 --> 00:04:13,920 How does it have a dramatic effect on the cell as a whole? 80 00:04:13,920 --> 00:04:17,250 How does the timing and action of this effect 81 00:04:17,250 --> 00:04:19,600 occur so rapidly? 82 00:04:19,600 --> 00:04:23,400 So we talked last time about different types 83 00:04:23,400 --> 00:04:26,160 of signals, those that mostly occur 84 00:04:26,160 --> 00:04:29,880 in the cytoplasm of the cell with signals that 85 00:04:29,880 --> 00:04:33,600 are able to diffuse across the plasma membrane and bind 86 00:04:33,600 --> 00:04:37,560 to an intracellular receptor and then cause an action. 87 00:04:37,560 --> 00:04:40,560 But really, the most important ones for today 88 00:04:40,560 --> 00:04:43,680 are going to be the types of receptors 89 00:04:43,680 --> 00:04:44,970 that span the membrane. 90 00:04:55,450 --> 00:04:59,040 And the reason why these are much more significant, 91 00:04:59,040 --> 00:05:02,130 they're more in number, they're more predominant, 92 00:05:02,130 --> 00:05:05,100 is if you have a membrane spanning receptor, 93 00:05:05,100 --> 00:05:07,470 you have the opportunity to use signals 94 00:05:07,470 --> 00:05:09,570 of very, very different types. 95 00:05:09,570 --> 00:05:14,050 Small polar molecules, small proteins, lipids, amino acids, 96 00:05:14,050 --> 00:05:15,220 carbohydrates. 97 00:05:15,220 --> 00:05:17,490 You've got a much larger range of signals 98 00:05:17,490 --> 00:05:20,940 than you could possibly have if you restricted yourselves 99 00:05:20,940 --> 00:05:24,360 to the type of signals that can cross the cell membrane. 100 00:05:24,360 --> 00:05:28,470 Those are very limited to non-polar small molecules that 101 00:05:28,470 --> 00:05:30,190 can get across the membrane. 102 00:05:30,190 --> 00:05:32,670 The more dominant types of signals 103 00:05:32,670 --> 00:05:35,910 are going to be the ones that are outside the cell. 104 00:05:35,910 --> 00:05:38,130 They arrive at a cell surface. 105 00:05:38,130 --> 00:05:43,860 They bind to a receptor that is transmembrane and transduce 106 00:05:43,860 --> 00:05:47,040 a signal from the outside to the inside. 107 00:05:47,040 --> 00:05:56,000 So that's an important term here, 108 00:05:56,000 --> 00:05:59,690 the process of transducing external information 109 00:05:59,690 --> 00:06:01,270 to internal information. 110 00:06:01,270 --> 00:06:03,440 So when we started the course, we 111 00:06:03,440 --> 00:06:05,570 were really thinking about laying down 112 00:06:05,570 --> 00:06:09,470 the cellular membrane as an encased environment 113 00:06:09,470 --> 00:06:12,500 where things could occur at high concentrations. 114 00:06:12,500 --> 00:06:14,450 You could set up systems that were 115 00:06:14,450 --> 00:06:16,100 functional within membranes. 116 00:06:16,100 --> 00:06:20,060 But in doing that within a membrane encased area, 117 00:06:20,060 --> 00:06:22,850 in doing that, you've built a formidable barrier 118 00:06:22,850 --> 00:06:24,150 around the cell. 119 00:06:24,150 --> 00:06:26,440 So the types of receptors that we'll talk about 120 00:06:26,440 --> 00:06:29,960 are those that have adapted to take this external information 121 00:06:29,960 --> 00:06:34,290 into the cell and then have a cellular consequence occur. 122 00:06:34,290 --> 00:06:38,360 So I'm going to talk to you about two specific types 123 00:06:38,360 --> 00:06:44,330 of cellular switches, and they're 124 00:06:44,330 --> 00:06:45,950 going to be intracellular. 125 00:06:53,680 --> 00:06:58,110 And we're going to be referring back to these because they're 126 00:06:58,110 --> 00:07:02,680 going to be important as we dissect a signaling pathway. 127 00:07:02,680 --> 00:07:06,240 So the first type-- so stand aside a moment. 128 00:07:06,240 --> 00:07:09,210 Put aside a moment the actual process 129 00:07:09,210 --> 00:07:12,330 of a signal binding the response, 130 00:07:12,330 --> 00:07:14,910 both biochemical and biological. 131 00:07:14,910 --> 00:07:17,520 Let's look first at the molecular detail 132 00:07:17,520 --> 00:07:19,680 of these switches and see how they 133 00:07:19,680 --> 00:07:22,060 are adapted to their function. 134 00:07:22,060 --> 00:07:24,210 And the first type of cellular switch 135 00:07:24,210 --> 00:07:25,965 are what are known as G proteins. 136 00:07:29,950 --> 00:07:34,480 The G is because they bind guanidine nucleotides. 137 00:07:34,480 --> 00:07:36,890 So that's why they're called G proteins. 138 00:07:36,890 --> 00:07:46,560 They're small proteins that bind GDP or GTP. 139 00:07:46,560 --> 00:07:56,340 So this is the guanine nucleotide 140 00:07:56,340 --> 00:08:01,590 that has either two phosphates, guanidine diphosphate 141 00:08:01,590 --> 00:08:04,820 or triphosphate. 142 00:08:04,820 --> 00:08:08,880 So there may or may not be a third phosphate here. 143 00:08:08,880 --> 00:08:10,800 And the G proteins bind them. 144 00:08:10,800 --> 00:08:12,870 And the dynamics of the situation 145 00:08:12,870 --> 00:08:20,880 are that when the G proteins are bound to GDP, 146 00:08:20,880 --> 00:08:22,380 they are inactive. 147 00:08:25,470 --> 00:08:27,690 The switch is off. 148 00:08:27,690 --> 00:08:29,760 There's an aspect of the structure, 149 00:08:29,760 --> 00:08:33,330 and it's very dependent on how many phosphates there 150 00:08:33,330 --> 00:08:34,830 are in this structure. 151 00:08:34,830 --> 00:08:38,250 But it's when it's bound to the diphosphate variant 152 00:08:38,250 --> 00:08:44,130 of the nucleotide, then it's an off switch. 153 00:08:44,130 --> 00:08:53,355 And when it's bound to GTP, it's an on switch and it's active. 154 00:08:56,230 --> 00:08:59,970 So this is the molecular basis of one of the switches. 155 00:08:59,970 --> 00:09:03,870 It relies on the shape, the conformational dynamics 156 00:09:03,870 --> 00:09:06,160 of these small G proteins. 157 00:09:06,160 --> 00:09:08,070 And that shape is quite different 158 00:09:08,070 --> 00:09:12,480 if it's bound to guanidine diphosphate or triphosphate. 159 00:09:12,480 --> 00:09:13,980 And we'll take a look in a moment 160 00:09:13,980 --> 00:09:16,710 at how the structure, the shape, changes. 161 00:09:16,710 --> 00:09:20,700 The shape shifts upon binding the triphosphate analog. 162 00:09:20,700 --> 00:09:24,390 So this is a dynamic interconversion. 163 00:09:24,390 --> 00:09:27,330 When the GTP is hydrolyzed, you go back 164 00:09:27,330 --> 00:09:30,420 to the GDP-bound state, the off state. 165 00:09:30,420 --> 00:09:33,090 And there are a variety of proteins 166 00:09:33,090 --> 00:09:35,940 that actually help these processes, which we 167 00:09:35,940 --> 00:09:38,260 won't talk about in any detail. 168 00:09:38,260 --> 00:09:40,320 The main thing that you want to remember 169 00:09:40,320 --> 00:09:43,410 is that when the G proteins are bound to GTP, 170 00:09:43,410 --> 00:09:44,700 they're in a non-state. 171 00:09:44,700 --> 00:09:46,940 GDP, they're in an off state. 172 00:09:46,940 --> 00:09:49,320 And that's shown in this cartoon. 173 00:09:49,320 --> 00:09:53,610 And here I should be able to, if all goes according to plan, 174 00:09:53,610 --> 00:10:00,100 show you the structure of a GTP analog bound to a G protein. 175 00:10:00,100 --> 00:10:02,190 So lets-- this little guy is twirling around. 176 00:10:02,190 --> 00:10:03,720 He's settled down a little bit. 177 00:10:03,720 --> 00:10:05,130 The key thing you want to look at 178 00:10:05,130 --> 00:10:08,520 is where it's magenta and cyan, the structure 179 00:10:08,520 --> 00:10:13,620 of the GDP and GTP-bound G protein are very, very similar. 180 00:10:13,620 --> 00:10:16,720 But big changes happen in the yellow, 181 00:10:16,720 --> 00:10:20,400 which is the GDP-bound form, and the red, 182 00:10:20,400 --> 00:10:22,050 which is the GT-bound form. 183 00:10:22,050 --> 00:10:26,220 Let me go back again so you can see that one more time. 184 00:10:26,220 --> 00:10:33,390 So in the GTP-bound form, a portion of the protein 185 00:10:33,390 --> 00:10:39,720 swings around and binds to that third phosphate on the GTP 186 00:10:39,720 --> 00:10:42,390 and forms a different shape to the structure. 187 00:10:42,390 --> 00:10:44,280 And that's a dynamic change that's 188 00:10:44,280 --> 00:10:46,260 responsible for activation. 189 00:10:46,260 --> 00:10:49,200 When it's just GDP, that's shorter. 190 00:10:49,200 --> 00:10:51,690 There's nothing for that red arm to bind. 191 00:10:51,690 --> 00:10:54,300 And so it's much more of a floppy structure. 192 00:10:54,300 --> 00:10:57,630 What I want you to notice in that little yellow portion 193 00:10:57,630 --> 00:11:01,530 in the GDP-bound form, you actually 194 00:11:01,530 --> 00:11:04,470 don't really see where the rest of the protein is. 195 00:11:04,470 --> 00:11:07,160 This is because this is a crystal structure. 196 00:11:07,160 --> 00:11:10,710 And in the crystal structure, when things are very mobile, 197 00:11:10,710 --> 00:11:12,930 you can't even see electron density. 198 00:11:12,930 --> 00:11:14,880 It's as if the part of the protein 199 00:11:14,880 --> 00:11:17,670 isn't there because it is so dynamic. 200 00:11:17,670 --> 00:11:20,160 It's only in the GTP-bound form it 201 00:11:20,160 --> 00:11:22,650 forms this tight, compact structure 202 00:11:22,650 --> 00:11:26,080 that represents a switch that has been turned on. 203 00:11:26,080 --> 00:11:28,185 Does that make sense to everybody? 204 00:11:28,185 --> 00:11:29,310 Is everyone good with that? 205 00:11:29,310 --> 00:11:33,180 So just that change, that extra phosphate reaching further 206 00:11:33,180 --> 00:11:35,760 to the protein and making an interaction 207 00:11:35,760 --> 00:11:38,790 with the protein itself, makes the difference 208 00:11:38,790 --> 00:11:43,570 in the dynamics of the G protein and the activity here. 209 00:11:43,570 --> 00:11:48,060 Now, there are different types of G proteins. 210 00:11:48,060 --> 00:11:52,540 And you'll see both types reflected in this lecture. 211 00:11:52,540 --> 00:12:02,550 There are small G proteins, and they are monomeric. 212 00:12:08,390 --> 00:12:11,730 And then there are slightly more complicated G proteins 213 00:12:11,730 --> 00:12:12,930 that are trimeric. 214 00:12:12,930 --> 00:12:15,630 They have a heterotrimeric, structure. 215 00:12:15,630 --> 00:12:17,880 So they have quaternary structure 216 00:12:17,880 --> 00:12:20,340 where you have three different proteins 217 00:12:20,340 --> 00:12:22,798 as part of the complex. 218 00:12:22,798 --> 00:12:24,090 So the other ones are trimeric. 219 00:12:28,490 --> 00:12:35,610 And the G protein actually comprises three subunits 220 00:12:35,610 --> 00:12:37,670 where one of them is the important one that 221 00:12:37,670 --> 00:12:40,610 binds GDP or GTP. 222 00:12:40,610 --> 00:12:42,650 But they are a little bit more complicated. 223 00:12:42,650 --> 00:12:44,870 And in the first example when I talk 224 00:12:44,870 --> 00:12:47,180 about a particular response to adrenaline, 225 00:12:47,180 --> 00:12:50,130 we're going to see the trimeric G proteins. 226 00:12:50,130 --> 00:12:52,010 And because they're trimeric, that 227 00:12:52,010 --> 00:12:55,430 means there's three subunits. 228 00:12:55,430 --> 00:12:59,000 And the convention is that they get the Greek lettering system. 229 00:12:59,000 --> 00:13:06,133 So they are the alpha, beta, and gamma subunits. 230 00:13:06,133 --> 00:13:07,300 They've each got their name. 231 00:13:07,300 --> 00:13:10,970 They're three independent polypeptide chains. 232 00:13:10,970 --> 00:13:17,750 And it's actually the alpha subunit that binds GDP or GTP. 233 00:13:17,750 --> 00:13:22,450 So that's the formulation of one of the types of switches 234 00:13:22,450 --> 00:13:25,550 that we're going to see when we start to look at a pathway. 235 00:13:25,550 --> 00:13:27,290 What do you need to remember here, 236 00:13:27,290 --> 00:13:32,230 you need to focus on the fact that in one state, 237 00:13:32,230 --> 00:13:34,110 the protein is in an off state. 238 00:13:34,110 --> 00:13:37,700 It doesn't kick off a signaling pathway. 239 00:13:37,700 --> 00:13:42,670 But in the other state, the protein 240 00:13:42,670 --> 00:13:46,550 is a different shape because of binding a loop-- 241 00:13:46,550 --> 00:13:48,920 let's just make this a little longer-- 242 00:13:48,920 --> 00:13:51,280 to that phosphate that's negatively 243 00:13:51,280 --> 00:13:53,210 charged to the protein. 244 00:13:53,210 --> 00:13:55,160 So that's an on state. 245 00:13:55,160 --> 00:13:58,270 And they are very definitive types of structures. 246 00:13:58,270 --> 00:14:08,790 Now, both of these proteins are intracellular, 247 00:14:08,790 --> 00:14:12,900 which means they're part of the response once a signal reaches 248 00:14:12,900 --> 00:14:13,680 a cell. 249 00:14:13,680 --> 00:14:15,180 They are things that change. 250 00:14:15,180 --> 00:14:20,040 And they're what the signal gets transduced to, the G proteins. 251 00:14:20,040 --> 00:14:23,520 Now, there's another type of intracellular switch 252 00:14:23,520 --> 00:14:26,940 which is used very, very frequently in nature. 253 00:14:26,940 --> 00:14:29,910 And in fact, it crosses, permeates, 254 00:14:29,910 --> 00:14:33,210 through all kinds of cellular processes. 255 00:14:33,210 --> 00:14:34,950 And this is phosphorylation. 256 00:14:34,950 --> 00:14:38,950 So here are the G proteins, which is one. 257 00:14:38,950 --> 00:14:43,590 And the other one is phosphorylation. 258 00:14:46,810 --> 00:14:48,135 I don't like that chalk. 259 00:14:54,060 --> 00:14:57,750 OK, now remember, we talked about reactions 260 00:14:57,750 --> 00:15:00,540 of proteins that alter their behavior, 261 00:15:00,540 --> 00:15:02,700 their properties, their dynamics. 262 00:15:02,700 --> 00:15:05,730 So protein phosphorylation, remember, 263 00:15:05,730 --> 00:15:10,200 is a post-translational modification, a PTM. 264 00:15:10,200 --> 00:15:12,510 It's something that happens to a protein 265 00:15:12,510 --> 00:15:16,200 after it has been translated and folded. 266 00:15:16,200 --> 00:15:19,610 And the PTMs in a phosphorylation 267 00:15:19,610 --> 00:15:29,420 involve amino acids that have OH groups. 268 00:15:34,500 --> 00:15:37,440 So that's the structure of tyrosine 269 00:15:37,440 --> 00:15:39,780 where the squiggles represent the rest of the protein. 270 00:15:44,700 --> 00:15:53,120 And on phosphorylation, we append a phosphate group-- 271 00:15:53,120 --> 00:15:58,140 whoops, a minus, a minus-- 272 00:15:58,140 --> 00:16:01,890 to the oxygen on tyrosine on the side chain. 273 00:16:05,990 --> 00:16:07,900 So actually, it looks pretty different. 274 00:16:07,900 --> 00:16:10,280 It behaves pretty differently. 275 00:16:10,280 --> 00:16:13,730 There are two other residues in eukaryotic cells 276 00:16:13,730 --> 00:16:16,060 that are commonly phosphorylated. 277 00:16:16,060 --> 00:16:19,450 There are the other two that include OH groups. 278 00:16:19,450 --> 00:16:31,180 So they are serine and the third one-- 279 00:16:33,690 --> 00:16:38,760 as I run out of space, but you get the general message-- 280 00:16:38,760 --> 00:16:41,300 and threonine. 281 00:16:41,300 --> 00:16:45,070 So these are the three amino acids that commonly 282 00:16:45,070 --> 00:16:47,320 get a phosphate group attached. 283 00:16:47,320 --> 00:16:49,600 And they change their properties. 284 00:16:49,600 --> 00:16:51,080 They are called kinases. 285 00:16:51,080 --> 00:16:54,610 Kinase, the root of the word is actually "to change." 286 00:16:54,610 --> 00:16:58,140 So the enzymes that catalyze this change 287 00:16:58,140 --> 00:16:59,345 are known as kinases. 288 00:17:02,590 --> 00:17:06,780 And in contrast to the G proteins that use GTP, 289 00:17:06,780 --> 00:17:12,010 the kinases most commonly use ATP to give up a phosphate 290 00:17:12,010 --> 00:17:13,900 to phosphorylate the protein. 291 00:17:13,900 --> 00:17:18,400 So there's another substrate in this reaction is ATP. 292 00:17:18,400 --> 00:17:21,339 Now, when we look at this structure, 293 00:17:21,339 --> 00:17:22,839 there's two or three things I really 294 00:17:22,839 --> 00:17:26,760 want to call your attention to. 295 00:17:26,760 --> 00:17:28,890 If we're dealing with this kind of switch, 296 00:17:28,890 --> 00:17:33,990 we can go back to the off state by chopping up the GTP 297 00:17:33,990 --> 00:17:35,550 and making it GDP again. 298 00:17:35,550 --> 00:17:38,250 So that's how to turn the light back off. 299 00:17:38,250 --> 00:17:40,920 In the case of the kinases, we've 300 00:17:40,920 --> 00:17:45,030 got to do something to go back from this state 301 00:17:45,030 --> 00:17:48,900 to the non-modified state to turn the light switch off. 302 00:17:48,900 --> 00:17:53,560 So for every transformation in the cell that involves kinase, 303 00:17:53,560 --> 00:17:58,230 there is a corresponding set of enzymes 304 00:17:58,230 --> 00:18:01,320 that reverse the reaction called a phosphatase. 305 00:18:01,320 --> 00:18:03,270 It takes that group off again. 306 00:18:03,270 --> 00:18:04,660 So let me write that down here. 307 00:18:09,200 --> 00:18:11,370 Now, phosphate is used a lot. 308 00:18:11,370 --> 00:18:15,110 So this is a phosphoprotein phosphatase. 309 00:18:15,110 --> 00:18:17,420 So kinase puts the phosphate on. 310 00:18:17,420 --> 00:18:20,780 The phosphoprotein phosphatase takes the phosphate off. 311 00:18:20,780 --> 00:18:23,300 There are three types of amino acids 312 00:18:23,300 --> 00:18:27,170 that get most commonly modified in our cells-- 313 00:18:27,170 --> 00:18:30,130 tyrosine, serine, and threonine. 314 00:18:30,130 --> 00:18:32,660 And one of the ones that forms an important part 315 00:18:32,660 --> 00:18:35,450 of an extracellular signaling mechanism 316 00:18:35,450 --> 00:18:37,850 are the tyrosine kinases. 317 00:18:37,850 --> 00:18:43,670 And we'll delve into them in a little bit, not the section I'm 318 00:18:43,670 --> 00:18:45,050 going to cover now, but later. 319 00:18:45,050 --> 00:18:48,400 Because tyrosine, various kinases 320 00:18:48,400 --> 00:18:50,810 come in a lot of different flavors. 321 00:18:50,810 --> 00:18:52,490 The common flavors are whether you 322 00:18:52,490 --> 00:18:55,670 modify tyrosine or threonine/serine, 323 00:18:55,670 --> 00:18:57,950 because these are more similar to each other, 324 00:18:57,950 --> 00:18:59,090 and this guy is different. 325 00:18:59,090 --> 00:19:01,200 But we'll get to that later. 326 00:19:01,200 --> 00:19:10,010 So in the cell, we have about 20,000 genes 327 00:19:10,010 --> 00:19:18,380 that encode proteins, encoding genes. 328 00:19:21,200 --> 00:19:26,120 All right, 515 of those are kinases. 329 00:19:26,120 --> 00:19:29,640 That's a pretty big chunk of the genome you've got to accept. 330 00:19:29,640 --> 00:19:36,340 So in excess of 500 kinases. 331 00:19:36,340 --> 00:19:40,030 Specifically, protein kinases, the ones that modify. 332 00:19:40,030 --> 00:19:43,270 So there's a big hunk of the genome dedicated 333 00:19:43,270 --> 00:19:45,280 to this kind of activity. 334 00:19:45,280 --> 00:19:48,640 And there's a dynamic because there's also 335 00:19:48,640 --> 00:19:50,975 about 100 phosphatases. 336 00:19:50,975 --> 00:19:52,600 They are a little bit more promiscuous. 337 00:19:52,600 --> 00:19:54,610 You don't need so many of them. 338 00:19:54,610 --> 00:19:58,390 But a large sort of component of the genome 339 00:19:58,390 --> 00:20:02,590 is responsible for phosphorylating proteins 340 00:20:02,590 --> 00:20:05,380 and dephosphorylating phosphoproteins. 341 00:20:05,380 --> 00:20:08,815 And that part of the genome has its own special name. 342 00:20:12,920 --> 00:20:16,410 And it is called the kinome. 343 00:20:16,410 --> 00:20:18,460 I hope that's not too small in there. 344 00:20:18,460 --> 00:20:21,480 So if we're describing all the enzymes 345 00:20:21,480 --> 00:20:24,360 in the genome that catalyze phosphorylation, 346 00:20:24,360 --> 00:20:27,420 we would call it the kinome as a collective set. 347 00:20:27,420 --> 00:20:29,370 Because it's the set of kinases. 348 00:20:29,370 --> 00:20:32,640 And you'll hear that term quite commonly. 349 00:20:32,640 --> 00:20:34,650 And the kinome is really important 350 00:20:34,650 --> 00:20:38,130 and represents major, major therapeutic targets. 351 00:20:38,130 --> 00:20:39,930 Because it's when kinases go wrong 352 00:20:39,930 --> 00:20:42,610 that we have physiological defects. 353 00:20:42,610 --> 00:20:45,060 So let's just go back to this. 354 00:20:45,060 --> 00:20:49,050 You can see we've got the kinase and the phosphatase. 355 00:20:49,050 --> 00:20:52,830 The donor for phosphorylation is ATP. 356 00:20:52,830 --> 00:20:54,690 And it is the gamma phosphate that's 357 00:20:54,690 --> 00:20:57,930 transferred to the protein to switch it from the off state 358 00:20:57,930 --> 00:20:59,460 to the on state. 359 00:20:59,460 --> 00:21:02,280 It is a post-translational modification, 360 00:21:02,280 --> 00:21:05,700 meaning it occurs on a protein after the protein has 361 00:21:05,700 --> 00:21:07,550 been fully translated. 362 00:21:07,550 --> 00:21:10,340 There are a few cotranslational PTMs. 363 00:21:10,340 --> 00:21:13,530 That seems like a bit of an oxymoron, 364 00:21:13,530 --> 00:21:16,050 cotranslational modifications. 365 00:21:16,050 --> 00:21:17,960 But phosphorylation isn't one of them. 366 00:21:17,960 --> 00:21:19,050 Glycosylation is. 367 00:21:19,050 --> 00:21:21,480 And we won't go into those in any detail 368 00:21:21,480 --> 00:21:23,700 even though it breaks my heart not to go into those. 369 00:21:23,700 --> 00:21:33,850 But OK, all right, so now I want to first of all introduce you 370 00:21:33,850 --> 00:21:38,420 to a paradigm for signaling as opposed to really go 371 00:21:38,420 --> 00:21:40,840 into what's happening. 372 00:21:40,840 --> 00:21:42,820 And so one of the first paradigms 373 00:21:42,820 --> 00:21:46,810 is a situation where you have a cell. 374 00:21:46,810 --> 00:21:50,080 In the plasma membrane of that cell is a receptor. 375 00:21:50,080 --> 00:21:52,000 And it gets hit with a signal. 376 00:21:52,000 --> 00:21:55,120 So a signaling paradigm is that a molecule 377 00:21:55,120 --> 00:21:59,920 from outside the cell binds to something that's transmembrane. 378 00:21:59,920 --> 00:22:02,410 And then you start getting signal transduction 379 00:22:02,410 --> 00:22:03,670 through a pathway. 380 00:22:03,670 --> 00:22:08,177 So any extracellular signal could be game in this process. 381 00:22:08,177 --> 00:22:09,760 And then there's a sequence of events. 382 00:22:09,760 --> 00:22:12,460 Upon signal binding, there's a sequence 383 00:22:12,460 --> 00:22:16,660 of changes that ends you up with a final output. 384 00:22:16,660 --> 00:22:18,790 And generally, signaling pathways 385 00:22:18,790 --> 00:22:20,830 go through a number of steps where 386 00:22:20,830 --> 00:22:25,190 there is the opportunity for the amplification of a signal. 387 00:22:25,190 --> 00:22:27,610 So I talked to you last about time 388 00:22:27,610 --> 00:22:30,460 about some of the hallmarks of signaling. 389 00:22:30,460 --> 00:22:36,130 Specificity defines how accurately that extracellular 390 00:22:36,130 --> 00:22:38,350 signal binds to the receptor. 391 00:22:38,350 --> 00:22:42,160 But amplification really refers to how 392 00:22:42,160 --> 00:22:45,160 signals get bigger and bigger through certain steps 393 00:22:45,160 --> 00:22:47,260 in a signaling pathway in order to have 394 00:22:47,260 --> 00:22:51,190 a big impact in the cell, not just a single event going 395 00:22:51,190 --> 00:22:54,210 through a single pathway one molecule at a time. 396 00:22:54,210 --> 00:22:56,530 And we'll see that in the example that I show you. 397 00:23:00,040 --> 00:23:04,120 And then oftentimes when we look at signaling pathways, 398 00:23:04,120 --> 00:23:06,430 we care a great deal about what's 399 00:23:06,430 --> 00:23:09,490 the first response upon the signal hitting 400 00:23:09,490 --> 00:23:11,330 the outside of the cell. 401 00:23:11,330 --> 00:23:16,570 So in many signaling pathways, this could be a protein. 402 00:23:16,570 --> 00:23:21,100 The receptor could be a protein that is bound to a G protein. 403 00:23:21,100 --> 00:23:22,900 And that would be the first responder 404 00:23:22,900 --> 00:23:25,510 through the pathway that really triggers off 405 00:23:25,510 --> 00:23:27,250 the cellular events. 406 00:23:27,250 --> 00:23:30,520 OK, and now, so what I want to talk about now, is the-- 407 00:23:30,520 --> 00:23:31,630 I always get this wrong. 408 00:23:31,630 --> 00:23:36,520 I always thought it was flight or fight or fight or whatever, 409 00:23:36,520 --> 00:23:37,150 fright. 410 00:23:37,150 --> 00:23:40,420 I always thought it was flight or fright, but it's not. 411 00:23:40,420 --> 00:23:44,690 The response is actually the fight or flight. 412 00:23:44,690 --> 00:23:48,160 So let's set this up to understand 413 00:23:48,160 --> 00:23:51,190 why this is such a great manifestation 414 00:23:51,190 --> 00:23:54,340 of a cellular signaling response. 415 00:23:54,340 --> 00:23:56,920 Because it includes a lot of the hallmarks 416 00:23:56,920 --> 00:24:00,740 that are really characteristic of the cellular response. 417 00:24:00,740 --> 00:24:08,860 So this response involves a cellular receptor 418 00:24:08,860 --> 00:24:20,530 that is called a G protein coupled receptor. 419 00:24:23,950 --> 00:24:26,110 We saw a little bit about them last time. 420 00:24:26,110 --> 00:24:31,970 They are always called GPCRs for short. 421 00:24:31,970 --> 00:24:34,240 And what that term means is that it's 422 00:24:34,240 --> 00:24:38,570 a receptor that is linked in some way to a G protein. 423 00:24:38,570 --> 00:24:50,695 So it could be coupled to a monomeric or a trimeric G 424 00:24:50,695 --> 00:24:51,195 protein. 425 00:24:55,030 --> 00:24:56,670 So don't confuse the two. 426 00:24:56,670 --> 00:24:58,260 One is the receptor. 427 00:24:58,260 --> 00:24:59,550 It's transmembrane. 428 00:24:59,550 --> 00:25:04,350 It's responsible for receiving signals and transducing them. 429 00:25:04,350 --> 00:25:07,020 The first responder is the G protein 430 00:25:07,020 --> 00:25:12,120 that changes from a GDP-bound stage to a GTP-bound state. 431 00:25:12,120 --> 00:25:14,400 And in the one that we're going to talk about, 432 00:25:14,400 --> 00:25:19,620 we're going to deal with a trimeric G protein in the fight 433 00:25:19,620 --> 00:25:21,480 or flight response. 434 00:25:21,480 --> 00:25:23,820 And what you see here is a cartoon 435 00:25:23,820 --> 00:25:25,950 of the players that are involved. 436 00:25:25,950 --> 00:25:28,560 So remember the G proteins? 437 00:25:28,560 --> 00:25:30,930 We talked about them briefly last time. 438 00:25:30,930 --> 00:25:36,210 They have seven transmembrane helices. 439 00:25:36,210 --> 00:25:38,770 They span the membrane. 440 00:25:38,770 --> 00:25:42,510 They have the N terminal outside. 441 00:25:42,510 --> 00:25:46,170 1, 2, 3, 4, 5, 6, 7. 442 00:25:46,170 --> 00:25:51,360 N terminus out, C terminus in. 443 00:25:51,360 --> 00:25:54,015 And each of these is a transmembrane helix. 444 00:25:58,100 --> 00:25:59,960 And this would be outside the cell. 445 00:25:59,960 --> 00:26:01,630 This would be in the cytoplasm. 446 00:26:04,780 --> 00:26:07,480 OK, and you can actually often look 447 00:26:07,480 --> 00:26:10,630 at the transmembrane protein and know its behavior. 448 00:26:10,630 --> 00:26:14,200 Because the width of these transmembrane 449 00:26:14,200 --> 00:26:21,310 helices often comes in at approximately 40 angstrom, 450 00:26:21,310 --> 00:26:23,060 which is the span of a membrane. 451 00:26:23,060 --> 00:26:25,690 You can sort of say, that looks like a transmembrane helix 452 00:26:25,690 --> 00:26:29,500 because it's exactly that dimension to cross a membrane. 453 00:26:29,500 --> 00:26:35,110 And the GPCRs in this case would bind 454 00:26:35,110 --> 00:26:39,100 to a ligand outside the cell and have 455 00:26:39,100 --> 00:26:41,660 a response inside the cell. 456 00:26:41,660 --> 00:26:44,110 So those seven transmembrane helices 457 00:26:44,110 --> 00:26:46,660 are responding to ligand binding. 458 00:26:46,660 --> 00:26:48,160 So let's take a look at this picture 459 00:26:48,160 --> 00:26:49,702 because it's almost impossible for me 460 00:26:49,702 --> 00:26:51,100 to get it onto the screen. 461 00:26:51,100 --> 00:26:54,610 So it binds to a trimeric G protein. 462 00:26:54,610 --> 00:26:57,760 Remember, I talked to you about the two different types. 463 00:26:57,760 --> 00:27:02,630 The trimeric has an alpha, beta, and gamma subunit quaternary 464 00:27:02,630 --> 00:27:03,790 structure. 465 00:27:03,790 --> 00:27:06,400 And they're shown here in different colors. 466 00:27:06,400 --> 00:27:08,770 The green is the alpha subunit. 467 00:27:12,010 --> 00:27:14,350 The red is the gamma subunit. 468 00:27:14,350 --> 00:27:18,130 And the yellow is the beta subunit. 469 00:27:18,130 --> 00:27:27,610 So what happens, when the ligand binds the G protein coupled 470 00:27:27,610 --> 00:27:30,760 receptor, there is a reorganization of those seven 471 00:27:30,760 --> 00:27:32,650 transmembrane helices. 472 00:27:32,650 --> 00:27:34,912 Last time, I identified them to you. 473 00:27:34,912 --> 00:27:36,370 When you look at a couple of these, 474 00:27:36,370 --> 00:27:39,760 they're actually fairly large loops 475 00:27:39,760 --> 00:27:41,580 that grab onto your ligand. 476 00:27:41,580 --> 00:27:45,130 And then that will translate conformational information 477 00:27:45,130 --> 00:27:47,560 through the membrane to the other side 478 00:27:47,560 --> 00:27:49,240 where the G proteins are sitting. 479 00:27:49,240 --> 00:27:52,120 And in this response, what happens 480 00:27:52,120 --> 00:27:58,300 is upon binding the ligand, the alpha subunit leaves the team 481 00:27:58,300 --> 00:28:04,180 and goes from their GDP-bound state to the GT-bound state. 482 00:28:04,180 --> 00:28:06,610 So it literally changes its state 483 00:28:06,610 --> 00:28:08,770 and changes its mode of association 484 00:28:08,770 --> 00:28:11,420 within the cell upon that action. 485 00:28:11,420 --> 00:28:14,310 So you can see how nicely we have transduced 486 00:28:14,310 --> 00:28:18,730 the ligand binding out here to a pretty discrete cellular event, 487 00:28:18,730 --> 00:28:22,618 turning on the switch of the G protein alpha subunit. 488 00:28:22,618 --> 00:28:23,910 Is everyone following me there? 489 00:28:23,910 --> 00:28:26,390 I know it sort of looks complicated to start with. 490 00:28:26,390 --> 00:28:28,600 But you'll see it in action. 491 00:28:28,600 --> 00:28:36,290 OK, so here are the cellular components of the response. 492 00:28:36,290 --> 00:28:38,650 So basically, this is the kind of response 493 00:28:38,650 --> 00:28:43,210 where if you get scared or you feel you're in harm's way, 494 00:28:43,210 --> 00:28:45,820 you will trigger this response in order 495 00:28:45,820 --> 00:28:51,040 to generate a lot of ATP in order that you can respond-- 496 00:28:51,040 --> 00:28:55,480 run away, hide, do something very active 497 00:28:55,480 --> 00:28:59,780 in order to rapidly respond to a threat of some kind. 498 00:28:59,780 --> 00:29:03,790 And this response is triggered by a small molecule. 499 00:29:03,790 --> 00:29:07,720 In this case, it's epinephrine or adrenaline. 500 00:29:07,720 --> 00:29:10,120 Different names on different sides of the Atlantic, 501 00:29:10,120 --> 00:29:12,040 but you all know what adrenaline is. 502 00:29:12,040 --> 00:29:15,520 And here's the structure of epinephrine or adrenaline. 503 00:29:15,520 --> 00:29:21,790 And it is the signal for the flight or fight response 504 00:29:21,790 --> 00:29:25,600 because it's the small molecule that binds to the extracellular 505 00:29:25,600 --> 00:29:28,930 surface of the receptor, changes its shape 506 00:29:28,930 --> 00:29:31,700 so that things can happen intracellularly. 507 00:29:31,700 --> 00:29:34,120 And it's just one small molecule. 508 00:29:34,120 --> 00:29:35,650 Normally that would be charged. 509 00:29:35,650 --> 00:29:37,940 It doesn't diffuse across the membrane. 510 00:29:37,940 --> 00:29:41,140 So it's stuck being on the outside of the membrane, OK? 511 00:29:41,140 --> 00:29:44,230 So this is the signal that triggers the response. 512 00:29:44,230 --> 00:29:49,330 So if you have to respond to a threat of some, 513 00:29:49,330 --> 00:29:54,220 kind you can't stop, sort of go to the fridge, get a big snack, 514 00:29:54,220 --> 00:29:57,310 eat it, digest all your food, and hope you're 515 00:29:57,310 --> 00:29:58,920 going to get energy quickly. 516 00:29:58,920 --> 00:30:00,820 What you've got to do is have a response 517 00:30:00,820 --> 00:30:04,300 where you can generate energy from your glycogen stores that 518 00:30:04,300 --> 00:30:05,440 are in the liver. 519 00:30:05,440 --> 00:30:10,240 So there is a signal comes from the adrenal region, which 520 00:30:10,240 --> 00:30:14,140 is the release of adrenaline that goes to the cell surface 521 00:30:14,140 --> 00:30:16,540 receptors to trigger the response. 522 00:30:16,540 --> 00:30:18,470 And what kind of signal would this be? 523 00:30:18,470 --> 00:30:22,300 Would it be paracrine, autocrine, exocrine? 524 00:30:22,300 --> 00:30:25,540 What kind of signal would that be? 525 00:30:25,540 --> 00:30:28,910 Sorry-- endocrine, paracrine, juxtacrine 526 00:30:28,910 --> 00:30:30,160 Do you remember last time? 527 00:30:30,160 --> 00:30:30,660 Yeah-- 528 00:30:30,660 --> 00:30:31,493 AUDIENCE: Endocrine. 529 00:30:31,493 --> 00:30:33,850 PROFESSOR: Endocrine, so it's a response that 530 00:30:33,850 --> 00:30:36,370 comes from the kidneys and goes to the liver. 531 00:30:36,370 --> 00:30:38,980 So it's going, it's traveling. 532 00:30:38,980 --> 00:30:41,020 Autocrine is self. 533 00:30:41,020 --> 00:30:42,570 Paracrine is near. 534 00:30:42,570 --> 00:30:44,320 Juxtacrine is cell contact. 535 00:30:44,320 --> 00:30:46,720 But any of these hormonal responses 536 00:30:46,720 --> 00:30:49,970 are pretty commonly endocrine responses. 537 00:30:49,970 --> 00:30:53,320 So what happens once the signal binds? 538 00:30:53,320 --> 00:30:57,490 So the specificity in this situation is that the G protein 539 00:30:57,490 --> 00:31:01,600 coupled receptor-- now shown in pink in very stylized form, 540 00:31:01,600 --> 00:31:05,290 but you can count those seven transmembrane domains-- 541 00:31:05,290 --> 00:31:11,290 will bind exclusively to this GPCR with high specificity. 542 00:31:11,290 --> 00:31:13,240 Another signal, another small molecule 543 00:31:13,240 --> 00:31:16,270 that looks like it won't bind because we have to have 544 00:31:16,270 --> 00:31:18,280 specificity for the signal. 545 00:31:18,280 --> 00:31:21,970 Upon that binding event, it will trigger a change 546 00:31:21,970 --> 00:31:23,320 within the cell. 547 00:31:23,320 --> 00:31:27,340 And that change within the cell is that the alpha subunit-- 548 00:31:27,340 --> 00:31:31,900 here you see alpha subunit, beta, gamma. 549 00:31:31,900 --> 00:31:33,740 They're all shown in green. 550 00:31:33,740 --> 00:31:36,550 The alpha subunit of the G protein, 551 00:31:36,550 --> 00:31:38,710 remember, I told you it was a trimeric G 552 00:31:38,710 --> 00:31:42,370 protein where the alpha subunit is the key player. 553 00:31:42,370 --> 00:31:49,660 The alpha subunit leaves the team and it exchanges its GDP-- 554 00:31:49,660 --> 00:31:51,430 that's its resting state, nothing's 555 00:31:51,430 --> 00:31:54,890 happening-- for GTP, which turns it on. 556 00:31:54,890 --> 00:31:56,960 So that's the first response. 557 00:31:56,960 --> 00:31:59,230 The G protein is responding to the signal 558 00:31:59,230 --> 00:32:02,260 from the outside of the cell through the auspices 559 00:32:02,260 --> 00:32:04,480 of the G protein coupled receptor 560 00:32:04,480 --> 00:32:07,910 to give a change within the cell that's a discrete change. 561 00:32:07,910 --> 00:32:10,000 OK, following me so far? 562 00:32:10,000 --> 00:32:13,150 So now what we need to do is trigger the remaining 563 00:32:13,150 --> 00:32:16,150 biochemical events that are going to get us out 564 00:32:16,150 --> 00:32:18,160 of this sticky situation where we 565 00:32:18,160 --> 00:32:20,660 need to produce a lot of ATP. 566 00:32:20,660 --> 00:32:24,190 So it turns out that the GTP-bound form 567 00:32:24,190 --> 00:32:28,480 of the alpha subunit can then bind to another enzyme. 568 00:32:28,480 --> 00:32:32,170 And that enzyme is adenylate cyclase. 569 00:32:32,170 --> 00:32:37,190 So we've bound, we've changed the GDP to GTP, 570 00:32:37,190 --> 00:32:39,760 there's a response, and we activate 571 00:32:39,760 --> 00:32:42,490 the enzyme known as AC, which you look up here, 572 00:32:42,490 --> 00:32:44,650 it's called adenylate cyclase. 573 00:32:44,650 --> 00:32:47,620 So this is a messenger within the cell that's 574 00:32:47,620 --> 00:32:50,290 now being generated as a response 575 00:32:50,290 --> 00:32:54,520 to the signal coming from outside through the GPCR 576 00:32:54,520 --> 00:32:57,320 to the alpha subunit of the G protein, 577 00:32:57,320 --> 00:33:02,110 which, then, in its GTP-bound state, binds adenylate cyclase. 578 00:33:02,110 --> 00:33:04,130 OK, is everyone with me? 579 00:33:04,130 --> 00:33:07,360 And once that is bound, adenylate cyclase 580 00:33:07,360 --> 00:33:09,310 can do its biochemistry. 581 00:33:09,310 --> 00:33:12,070 And the biochemistry that adenylate cyclase does 582 00:33:12,070 --> 00:33:13,390 is shown down here. 583 00:33:13,390 --> 00:33:15,000 Here's ATP. 584 00:33:15,000 --> 00:33:20,380 Adenylate cyclase cyclizes ATP. 585 00:33:20,380 --> 00:33:22,450 You lose two of the phosphates, and you 586 00:33:22,450 --> 00:33:25,900 get this molecule known as cyclic AMP, which 587 00:33:25,900 --> 00:33:28,960 is a messenger molecule that will propagate information 588 00:33:28,960 --> 00:33:31,660 through the cell. 589 00:33:31,660 --> 00:33:35,050 So now, the adenylate cyclase is activated 590 00:33:35,050 --> 00:33:38,380 because it's bound to the GTP-bound form 591 00:33:38,380 --> 00:33:41,020 of the alpha subunit. 592 00:33:41,020 --> 00:33:44,650 That means we can make a bunch of cyclic AMP. 593 00:33:44,650 --> 00:33:48,220 And cyclic AMP is what's known as a second messenger. 594 00:34:01,280 --> 00:34:03,910 And that often means it's a common messenger 595 00:34:03,910 --> 00:34:05,170 in a lot of pathways. 596 00:34:05,170 --> 00:34:08,989 It shows up quite frequently within the wiring of a pathway. 597 00:34:08,989 --> 00:34:14,659 And it acts locally to where the pathway is being processed. 598 00:34:14,659 --> 00:34:21,429 So once cyclic AMP is formed by adenylate cyclase, 599 00:34:21,429 --> 00:34:24,040 that then activates an enzyme. 600 00:34:24,040 --> 00:34:29,849 It activates protein kinase A. So PKA is a kinase. 601 00:34:34,650 --> 00:34:37,480 It's actually a serine threonine kinase. 602 00:34:47,050 --> 00:34:49,900 And that then results in certain proteins 603 00:34:49,900 --> 00:34:53,710 within the cell becoming phosphorylated to continue 604 00:34:53,710 --> 00:34:55,639 propagating our effect. 605 00:34:55,639 --> 00:35:00,070 So we have specificity by the adrenaline binding. 606 00:35:00,070 --> 00:35:04,670 We have amplification somewhere in this pathway. 607 00:35:04,670 --> 00:35:07,870 So I told you that many pathways go through steps where you 608 00:35:07,870 --> 00:35:09,910 start amplifying the signal. 609 00:35:09,910 --> 00:35:12,070 Where do you think is the first stage 610 00:35:12,070 --> 00:35:14,050 in this set of transformations that I've 611 00:35:14,050 --> 00:35:19,250 described to you where you start amplifying the information? 612 00:35:19,250 --> 00:35:24,140 Think about what each of the events comprises. 613 00:35:24,140 --> 00:35:26,540 Is this one binding to one in one event, 614 00:35:26,540 --> 00:35:29,960 or is it one binding to one and we get multiple events? 615 00:35:29,960 --> 00:35:33,950 Where is the first step of amplification that's essential? 616 00:35:33,950 --> 00:35:35,570 Because it wouldn't do us any good 617 00:35:35,570 --> 00:35:39,180 if we make one molecule of ATP at the end of the day. 618 00:35:39,180 --> 00:35:42,320 We've got to make dozens and dozens of molecules of ATP. 619 00:35:42,320 --> 00:35:46,920 What's the first event that could be an amplification? 620 00:35:46,920 --> 00:35:47,420 Over there-- 621 00:35:47,420 --> 00:35:49,785 AUDIENCE: Is it when [INAUDIBLE] 622 00:35:49,785 --> 00:35:52,160 PROFESSOR: Yeah, when it's made, yes. 623 00:35:52,160 --> 00:35:54,110 So this, let's go through them. 624 00:35:54,110 --> 00:35:56,240 One binds to one, great. 625 00:35:56,240 --> 00:35:59,180 Once one binds to one, one of these is released. 626 00:35:59,180 --> 00:36:01,280 It gets converted to one of these. 627 00:36:01,280 --> 00:36:05,400 Once one of these is made, adenylate cyclase is an enzyme, 628 00:36:05,400 --> 00:36:08,120 so it can make a bunch of cyclic AMP, 629 00:36:08,120 --> 00:36:10,850 which can then activate a bunch of protein kinase 630 00:36:10,850 --> 00:36:15,110 A, which can then phosphorylate a bunch of cellular proteins. 631 00:36:15,110 --> 00:36:17,535 So we've got an expansion of our response. 632 00:36:17,535 --> 00:36:19,910 All right, so everyone, does that make sense to everyone? 633 00:36:19,910 --> 00:36:22,490 So amplification is really important. 634 00:36:22,490 --> 00:36:25,010 Feedback is also important. 635 00:36:25,010 --> 00:36:28,730 If you ended up needing an EpiPen because you 636 00:36:28,730 --> 00:36:30,920 have an allergic response, you might 637 00:36:30,920 --> 00:36:33,050 remember that you've got the jitters forever 638 00:36:33,050 --> 00:36:36,080 because there's too much firing and action going on. 639 00:36:36,080 --> 00:36:38,420 But in the fight or flight response, 640 00:36:38,420 --> 00:36:40,730 there's feedback at a certain stage that 641 00:36:40,730 --> 00:36:43,340 slows down this entire process. 642 00:36:43,340 --> 00:36:46,130 And that feedback actually comes from an enzyme 643 00:36:46,130 --> 00:36:49,490 that chomps up the cyclic AMP to make it 644 00:36:49,490 --> 00:36:51,590 inactive as a second messenger. 645 00:36:51,590 --> 00:36:53,950 So there's feedback in this process. 646 00:36:53,950 --> 00:36:56,660 OK, now, what happens within the cell 647 00:36:56,660 --> 00:36:59,310 to get us that biological response? 648 00:36:59,310 --> 00:37:02,450 This is a sort of a shortened version of what's happening. 649 00:37:02,450 --> 00:37:04,310 So epinephrine binds. 650 00:37:04,310 --> 00:37:08,690 Here we are with the alpha subunit with cyclic AMP. 651 00:37:08,690 --> 00:37:10,700 For each one of these, you might make 652 00:37:10,700 --> 00:37:13,310 20 molecules of cyclic AMP. 653 00:37:13,310 --> 00:37:17,390 That would inactivate many, many PKAs. 654 00:37:17,390 --> 00:37:20,570 And then you go through a series of biochemical steps 655 00:37:20,570 --> 00:37:23,420 where different enzymes are activated 656 00:37:23,420 --> 00:37:28,970 with the overall goal of in the liver chewing up glycogen. OK, 657 00:37:28,970 --> 00:37:32,960 so glycogen is a pretty impenetrable polymer 658 00:37:32,960 --> 00:37:34,730 of carbohydrates. 659 00:37:34,730 --> 00:37:39,500 And you need several enzymes to start to break glycogen down 660 00:37:39,500 --> 00:37:42,090 to make glucose phosphate. 661 00:37:42,090 --> 00:37:48,050 And so these enzymes here of phosphorylase, B kinase, 662 00:37:48,050 --> 00:37:52,820 glycogen phosphorylase all end up converting glycogen 663 00:37:52,820 --> 00:37:54,440 into glucose 1 phosphate. 664 00:37:54,440 --> 00:37:58,790 So you access your liver stores of stored carbohydrate, which 665 00:37:58,790 --> 00:38:03,650 is in a polymeric form, to get a lot of glucose phosphate, which 666 00:38:03,650 --> 00:38:07,280 is then hydrolyzed to glucose, which then hits the blood 667 00:38:07,280 --> 00:38:08,180 system. 668 00:38:08,180 --> 00:38:11,570 And then you can deliver glucose to all the cells 669 00:38:11,570 --> 00:38:14,570 to go glycolysis and make ATP. 670 00:38:14,570 --> 00:38:16,835 And every glucose molecule, as you know, 671 00:38:16,835 --> 00:38:19,230 can really churn out ATP. 672 00:38:19,230 --> 00:38:21,380 So what we see in this process is 673 00:38:21,380 --> 00:38:24,500 going through the entire dynamics of the system 674 00:38:24,500 --> 00:38:28,760 where we've seen specificity, amplification, and feedback. 675 00:38:28,760 --> 00:38:32,234 Later on, I'll describe integration to you. 676 00:38:32,234 --> 00:38:34,730 OK, everyone following? 677 00:38:34,730 --> 00:38:38,690 The series of steps that go from a molecular messenger 678 00:38:38,690 --> 00:38:42,320 to biochemical steps to physiological, biological 679 00:38:42,320 --> 00:38:43,790 response. 680 00:38:43,790 --> 00:38:46,970 Now, I want to just emphasize one quick thing here. 681 00:38:46,970 --> 00:38:51,830 I've got a couple of slides I popped in of drug targets. 682 00:38:51,830 --> 00:38:55,220 About 45% are receptors in cells. 683 00:38:55,220 --> 00:39:00,440 25% of the entire drug targets are GPCRs. 684 00:39:00,440 --> 00:39:03,520 They respond to all kinds of signals-- 685 00:39:03,520 --> 00:39:07,370 amines, amino acids, lipids, little peptides, proteins, 686 00:39:07,370 --> 00:39:08,540 nucleotides-- 687 00:39:08,540 --> 00:39:11,360 all commonly going through the G protein 688 00:39:11,360 --> 00:39:14,870 coupled receptor to give you a similar phenomenon to what I've 689 00:39:14,870 --> 00:39:16,370 described to you. 690 00:39:16,370 --> 00:39:19,580 And what I think is particularly interesting-- 691 00:39:19,580 --> 00:39:21,950 I'm going to post all of these as slides. 692 00:39:21,950 --> 00:39:24,500 What I want you to see, this was quite a while 693 00:39:24,500 --> 00:39:28,580 back, but it just shows you so many of trademarked drugs that 694 00:39:28,580 --> 00:39:31,460 target different GPCRs-- 695 00:39:31,460 --> 00:39:34,610 they're shown here-- and what diseases they're 696 00:39:34,610 --> 00:39:38,120 used for to treat, and what's the generic name 697 00:39:38,120 --> 00:39:39,210 of those drugs. 698 00:39:39,210 --> 00:39:41,900 So you can see here many, many diseases 699 00:39:41,900 --> 00:39:45,380 have at the heart and soul of their problem 700 00:39:45,380 --> 00:39:46,370 different receptors. 701 00:39:46,370 --> 00:39:49,490 And these are all G protein coupled receptors 702 00:39:49,490 --> 00:39:51,820 that are treated with small molecules 703 00:39:51,820 --> 00:39:54,620 that bind to the receptor and often glue it 704 00:39:54,620 --> 00:39:57,200 in an inactive state so it can't, then, 705 00:39:57,200 --> 00:40:01,130 bind to an activating signal and have all the rest of the events 706 00:40:01,130 --> 00:40:02,780 occur. 707 00:40:02,780 --> 00:40:05,780 There are very few structures of the G protein 708 00:40:05,780 --> 00:40:08,460 coupled receptors, but there are some of them. 709 00:40:08,460 --> 00:40:12,050 So many of the target G protein coupled receptors 710 00:40:12,050 --> 00:40:14,960 can be modeled computationally. 711 00:40:14,960 --> 00:40:17,480 And then you can do a lot of work 712 00:40:17,480 --> 00:40:19,670 where you actually model the receptor 713 00:40:19,670 --> 00:40:22,490 in a membrane environment and start 714 00:40:22,490 --> 00:40:26,300 searching for drugs through computational approaches. 715 00:40:26,300 --> 00:40:28,700 And I thought a lot of you might be interested in this. 716 00:40:28,700 --> 00:40:31,160 Because this is a really strong axis 717 00:40:31,160 --> 00:40:33,910 where bioinformatics, confirmation, 718 00:40:33,910 --> 00:40:37,040 and advanced physics and molecular dynamics 719 00:40:37,040 --> 00:40:39,740 can be brought to bear on drug discovery 720 00:40:39,740 --> 00:40:42,680 when you don't have perfect molecular models 721 00:40:42,680 --> 00:40:44,820 of your targets. 722 00:40:44,820 --> 00:40:48,800 OK, so now we're going to move to a different kind of signal. 723 00:40:48,800 --> 00:40:52,580 We're going to talk about the receptor tyrosine kinases. 724 00:40:52,580 --> 00:40:57,860 All right, so in the receptor tyrosine kinase responses, 725 00:40:57,860 --> 00:41:01,070 we can often see very similar paradigms 726 00:41:01,070 --> 00:41:04,750 to what I've just shown you. 727 00:41:04,750 --> 00:41:08,090 But there is an important distinction. 728 00:41:08,090 --> 00:41:24,290 Receptor tyrosine kinases, we often call these RTKs. 729 00:41:24,290 --> 00:41:26,300 So that's their shorthand. 730 00:41:26,300 --> 00:41:30,830 So over here, I described to you different kinases. 731 00:41:30,830 --> 00:41:35,270 That we have kinases that modify threonine, serine, 732 00:41:35,270 --> 00:41:36,565 and tyrosine. 733 00:41:36,565 --> 00:41:41,870 The receptor tyrosine kinase is a subset of tyrosine kinases 734 00:41:41,870 --> 00:41:44,280 that form part of a receptor. 735 00:41:44,280 --> 00:41:47,360 So if you were to think about various kinases, 736 00:41:47,360 --> 00:41:51,500 you would have the serine/threonine, 737 00:41:51,500 --> 00:41:53,620 and you would have the tyrosine ones. 738 00:41:53,620 --> 00:41:56,150 But these would be differentiated 739 00:41:56,150 --> 00:42:03,280 into the ones that are part of a membrane protein, the RTKs, 740 00:42:03,280 --> 00:42:06,590 and then the ones that are soluble in the cytoplasm. 741 00:42:06,590 --> 00:42:09,380 And the serine/threonine ones are most commonly 742 00:42:09,380 --> 00:42:11,270 soluble in the cytoplasm. 743 00:42:11,270 --> 00:42:14,840 I'm going to focus on the receptor tyrosine kinases 744 00:42:14,840 --> 00:42:17,780 because they do slightly different activities when they 745 00:42:17,780 --> 00:42:20,390 signal relative to the GPCRs. 746 00:42:20,390 --> 00:42:23,060 So that's once again, this is a situation, 747 00:42:23,060 --> 00:42:26,540 another paradigm where you see a series of events. 748 00:42:26,540 --> 00:42:28,610 But with a number of the receptor tyrosine 749 00:42:28,610 --> 00:42:31,460 kinase pathways, the ultimate action 750 00:42:31,460 --> 00:42:34,070 ends up being in the nucleus where, 751 00:42:34,070 --> 00:42:36,620 as a result of an extracellular signal, 752 00:42:36,620 --> 00:42:40,460 you get a series of events that ends up with a protein being 753 00:42:40,460 --> 00:42:42,620 sent into the nucleus. 754 00:42:42,620 --> 00:42:45,890 And that protein may be a transcription factor that 755 00:42:45,890 --> 00:42:48,180 binds to a promoter region. 756 00:42:48,180 --> 00:42:53,220 And as a result of that, you'll get gene transcription occur. 757 00:42:53,220 --> 00:42:56,030 You'll transcribe a gene, make a messenger 758 00:42:56,030 --> 00:42:59,120 RNA that will leave the nucleus and cause action 759 00:42:59,120 --> 00:43:00,235 within the cell. 760 00:43:00,235 --> 00:43:01,610 So this is a little bit different 761 00:43:01,610 --> 00:43:06,620 than the other response that was mainly cytoplasmic. 762 00:43:06,620 --> 00:43:10,470 OK, so let's take a look at the receptor tyrosine kinases. 763 00:43:10,470 --> 00:43:13,440 Receptor tyrosine kinases are proteins 764 00:43:13,440 --> 00:43:16,800 that span the membrane but rather differently 765 00:43:16,800 --> 00:43:19,410 from the GPCRs. 766 00:43:19,410 --> 00:43:22,790 And they have a domain that's extracellular, just 767 00:43:22,790 --> 00:43:25,050 a single transmembrane domain. 768 00:43:25,050 --> 00:43:26,730 So this is out. 769 00:43:26,730 --> 00:43:27,840 This is in. 770 00:43:27,840 --> 00:43:31,170 And then they have an intracellular domain. 771 00:43:31,170 --> 00:43:33,705 This would be where the ligand binds. 772 00:43:37,710 --> 00:43:41,370 This would be how there's some kind of signal transducing. 773 00:43:41,370 --> 00:43:44,500 And this would be a kinase domain. 774 00:43:50,180 --> 00:43:52,410 OK, so how do we get the information in? 775 00:43:52,410 --> 00:43:57,260 When we saw the GPCRs, we saw the ability of those seven TMs 776 00:43:57,260 --> 00:44:00,830 to kind of reorganize and send information in the cell. 777 00:44:00,830 --> 00:44:03,050 With the receptor tyrosine kinases, 778 00:44:03,050 --> 00:44:04,580 it's kind of different. 779 00:44:04,580 --> 00:44:07,100 There are regions of the membrane where there 780 00:44:07,100 --> 00:44:09,140 are a lot of these proteins. 781 00:44:09,140 --> 00:44:13,120 They commonly bind small peptides and protein molecules. 782 00:44:13,120 --> 00:44:16,310 And when they're in their activated form, 783 00:44:16,310 --> 00:44:20,300 once the small protein binds, the receptor tyrosine kinase 784 00:44:20,300 --> 00:44:22,550 forms a dimeric structure. 785 00:44:22,550 --> 00:44:26,480 That is, two of these get together only 786 00:44:26,480 --> 00:44:27,600 upon ligand binding. 787 00:44:31,530 --> 00:44:36,510 They move together once there is a ligand bound. 788 00:44:36,510 --> 00:44:40,410 And then what happens is that the tyrosine kinase domains 789 00:44:40,410 --> 00:44:42,450 phosphorylate each other. 790 00:44:42,450 --> 00:44:46,050 And that's activation in the case of receptor tyrosine 791 00:44:46,050 --> 00:44:47,340 kinases. 792 00:44:47,340 --> 00:44:50,980 So when the small protein ligand is not around, 793 00:44:50,980 --> 00:44:52,620 this is a singleton. 794 00:44:52,620 --> 00:44:54,570 It doesn't work on itself. 795 00:44:54,570 --> 00:44:58,410 Once this ligand binds, interactions change. 796 00:44:58,410 --> 00:45:01,650 You get a dimeric structure where one kinase 797 00:45:01,650 --> 00:45:05,100 can phosphorylate what's called in trans 798 00:45:05,100 --> 00:45:06,540 the other kinase domains. 799 00:45:06,540 --> 00:45:09,283 So it's different from the GPCRs. 800 00:45:09,283 --> 00:45:10,950 It's got a different kind of feel to it, 801 00:45:10,950 --> 00:45:14,400 but it's still a dynamic transient signal. 802 00:45:14,400 --> 00:45:17,040 Let's take a look at this within a cell 803 00:45:17,040 --> 00:45:18,960 and see what kinds of responses-- 804 00:45:18,960 --> 00:45:21,840 and this is in response to EGF, which 805 00:45:21,840 --> 00:45:24,150 is epidermal growth factor. 806 00:45:24,150 --> 00:45:27,700 It's a cytokine that promotes cell division. 807 00:45:27,700 --> 00:45:31,410 So a lot happens with respect to the action of a cell, 808 00:45:31,410 --> 00:45:34,710 not to produce ATP, but now to respond 809 00:45:34,710 --> 00:45:38,580 by producing all the elements that enable cells to grow 810 00:45:38,580 --> 00:45:39,750 and proliferate. 811 00:45:39,750 --> 00:45:43,950 So the epidermal growth factor binds. 812 00:45:43,950 --> 00:45:45,930 You get dimerization. 813 00:45:45,930 --> 00:45:50,100 Upon that dimerization, the kinase domain in one 814 00:45:50,100 --> 00:45:52,020 structure-- in the blue one-- 815 00:45:52,020 --> 00:45:54,840 phosphorylates the other, and vice versa. 816 00:45:54,840 --> 00:45:58,610 They phosphorylate each other intermolecularly. 817 00:45:58,610 --> 00:46:02,130 Once that has happened, through the auspices of another protein 818 00:46:02,130 --> 00:46:04,590 I won't bother you with the name of, 819 00:46:04,590 --> 00:46:08,310 this phosphorylated intracellular RTK 820 00:46:08,310 --> 00:46:11,220 binds to a small G protein. 821 00:46:11,220 --> 00:46:14,130 In this case, it's a monomeric G protein, 822 00:46:14,130 --> 00:46:18,720 not one of the trimeric ones, a small one known as RAS. 823 00:46:18,720 --> 00:46:22,230 Once that binding event occurs, guess what? 824 00:46:22,230 --> 00:46:24,270 RAS gets activated. 825 00:46:24,270 --> 00:46:28,430 It's now binding GTP instead of GDP. 826 00:46:28,430 --> 00:46:31,590 And then it starts going through a sequence of events 827 00:46:31,590 --> 00:46:36,230 where there's a ton of controlled cellular 828 00:46:36,230 --> 00:46:38,250 phosphorylation events that result 829 00:46:38,250 --> 00:46:41,490 in moving a protein into the nucleus that helps 830 00:46:41,490 --> 00:46:44,550 form a transcription complex that results 831 00:46:44,550 --> 00:46:46,800 in cellular proliferation. 832 00:46:46,800 --> 00:46:49,050 Similar but different series of events. 833 00:46:49,050 --> 00:46:51,180 There's still amplification. 834 00:46:51,180 --> 00:46:52,680 There's still dynamics. 835 00:46:52,680 --> 00:46:56,000 And in this case, it's a lot of phosphorylation events. 836 00:46:56,000 --> 00:46:57,870 And what I want to sort of define 837 00:46:57,870 --> 00:47:01,260 for you is that many of these pathways 838 00:47:01,260 --> 00:47:03,790 are in trouble in disease states. 839 00:47:03,790 --> 00:47:07,770 Be it inflammation, neurodegeneration, or cancer, 840 00:47:07,770 --> 00:47:12,480 there is aberrant behavior of proteins within these pathways 841 00:47:12,480 --> 00:47:15,660 that cause them to go wrong, cause cells to proliferate out 842 00:47:15,660 --> 00:47:19,350 of control or undergo bad responses. 843 00:47:19,350 --> 00:47:21,390 And that is why these proteins end up 844 00:47:21,390 --> 00:47:24,930 being therapeutic targets like the G protein coupled 845 00:47:24,930 --> 00:47:25,590 receptors. 846 00:47:29,860 --> 00:47:33,260 OK, so we've seen the characteristics of signaling. 847 00:47:33,260 --> 00:47:34,550 We've seen a signal. 848 00:47:34,550 --> 00:47:36,380 We've seen amplification. 849 00:47:36,380 --> 00:47:38,000 We've seen responses. 850 00:47:38,000 --> 00:47:40,940 What I just want to quickly show you 851 00:47:40,940 --> 00:47:44,060 is an idea about integration. 852 00:47:44,060 --> 00:47:47,210 So here's an idea with two signaling pathways 853 00:47:47,210 --> 00:47:50,930 that sort of end up with the same signal outside where you 854 00:47:50,930 --> 00:47:54,560 integrate actions through two different signaling 855 00:47:54,560 --> 00:47:58,910 pathways to achieve a bigger, different kind of response. 856 00:47:58,910 --> 00:48:02,390 So that's that last hallmark of signaling pathways. 857 00:48:02,390 --> 00:48:05,480 It's not that every pathway is clean and straight. 858 00:48:05,480 --> 00:48:07,940 It has cross-talk with other pathways 859 00:48:07,940 --> 00:48:10,820 and you get amplified or different responses. 860 00:48:10,820 --> 00:48:12,680 Tremendously complicated. 861 00:48:12,680 --> 00:48:14,510 I want to give you one more term. 862 00:48:14,510 --> 00:48:16,460 And then I'll show one table. 863 00:48:16,460 --> 00:48:19,550 When these pathways go wrong, it's often 864 00:48:19,550 --> 00:48:22,160 because switches get stuck on. 865 00:48:22,160 --> 00:48:28,700 So for example, a G protein gets stuck in its GTP-bound state 866 00:48:28,700 --> 00:48:32,270 or doesn't even need GTP to be activated. 867 00:48:32,270 --> 00:48:35,630 Or a tyrosine kinase is stuck activated. 868 00:48:35,630 --> 00:48:47,210 And that's what's called constitutively active, 869 00:48:47,210 --> 00:48:49,505 basically meaning it's permanently on. 870 00:48:49,505 --> 00:48:53,450 So many of the diseases that are caused by mutations 871 00:48:53,450 --> 00:48:55,730 in your genome, not genetic diseases 872 00:48:55,730 --> 00:49:00,290 but mutations in your genes in some particular cells, 873 00:49:00,290 --> 00:49:02,540 end up with constitutive activation 874 00:49:02,540 --> 00:49:05,420 where you don't need a signal to have a response. 875 00:49:05,420 --> 00:49:08,930 And so for example, cells may proliferate out of control. 876 00:49:08,930 --> 00:49:13,490 So that is an important term to know and understand. 877 00:49:13,490 --> 00:49:16,220 Because constitutive activation basically 878 00:49:16,220 --> 00:49:19,640 means that a receptor may be active in the absence 879 00:49:19,640 --> 00:49:20,630 of a ligand. 880 00:49:20,630 --> 00:49:22,670 And I believe this is my last slide. 881 00:49:22,670 --> 00:49:24,800 I just wanted to leave you with this. 882 00:49:24,800 --> 00:49:27,620 When one thinks of GPCRs, there are 883 00:49:27,620 --> 00:49:30,110 tremendous therapeutic targets. 884 00:49:30,110 --> 00:49:33,430 The world of kinases is no less important. 885 00:49:33,430 --> 00:49:36,050 This scale is in billions of dollars 886 00:49:36,050 --> 00:49:41,270 spent on developing molecules that may be curative 887 00:49:41,270 --> 00:49:45,260 of diseases that involve dysregulated signaling. 888 00:49:45,260 --> 00:49:48,620 And what you see on this, I want to point out two things. 889 00:49:48,620 --> 00:49:52,480 Of course this thing stops working at the last minutes. 890 00:49:52,480 --> 00:49:56,210 But what I want to point out is this particular bar. 891 00:49:56,210 --> 00:49:58,370 This represents the billions of dollars 892 00:49:58,370 --> 00:50:01,120 spent on protein kinase inhibitors 893 00:50:01,120 --> 00:50:03,270 over a five-year period. 894 00:50:03,270 --> 00:50:05,840 And it's just escalating and escalating. 895 00:50:05,840 --> 00:50:09,200 Similarly, monoclonal antibodies are very important. 896 00:50:09,200 --> 00:50:12,860 But the small molecule drugs hold a real dominance. 897 00:50:12,860 --> 00:50:15,020 What do these drugs do? 898 00:50:15,020 --> 00:50:17,750 They enable you to have small molecules that 899 00:50:17,750 --> 00:50:20,990 can go into dysregulated signaling pathways 900 00:50:20,990 --> 00:50:25,550 and stop the activity somewhere in the pathway to avoid signals 901 00:50:25,550 --> 00:50:28,490 going constantly to the nucleus and turning things 902 00:50:28,490 --> 00:50:29,860 on all the time. 903 00:50:29,860 --> 00:50:33,935 So both of these types of functions in cellular signaling 904 00:50:33,935 --> 00:50:36,320 are ones you want to understand both 905 00:50:36,320 --> 00:50:40,100 from a biological perspective but from a medical perspective. 906 00:50:40,100 --> 00:50:41,650 OK--