1 00:00:04,688 --> 00:00:06,757 WELCOME TO THE PROTEOMICS 2 00:00:06,757 --> 00:00:09,560 INTEREST GROUP LECTURE SERIES. 3 00:00:09,560 --> 00:00:11,795 TODAY IS MY PLEASURE TO 4 00:00:11,795 --> 00:00:15,632 INTRODUCE OUR FIRST SPEAKER OF 5 00:00:15,632 --> 00:00:26,009 THE REGION 5 DR. RUTH 6 00:00:27,644 --> 00:00:29,079 HUTTENHAIN, SHE WORKS WITH 7 00:00:29,079 --> 00:00:30,681 COLLEAGUES WHERE SHE DID HER 8 00:00:30,681 --> 00:00:32,649 POST DOC AT THE UNIVERSITY OF 9 00:00:32,649 --> 00:00:34,051 CALIFORNIA SAN FRANCISCO WHERE 10 00:00:34,051 --> 00:00:35,085 HE STUDIED PROTEIN SIGNALING 11 00:00:35,085 --> 00:00:42,059 THAT WORKS IN HIV INFECTION. 12 00:00:42,059 --> 00:00:47,097 AND SHE PIONEERED A DRUG 13 00:00:47,097 --> 00:00:47,998 INTERACTION WITH SPATIAL 14 00:00:47,998 --> 00:00:50,400 RESOLUTION AND AFTER HER POST 15 00:00:50,400 --> 00:00:55,439 DOC SHE CONTINUED ADJUNCT 16 00:00:55,439 --> 00:00:58,275 ASSISTANT PROFESSOR AT UCSF AND 17 00:00:58,275 --> 00:01:01,044 RIGHT NOW SHE'S AT STANFORD AND 18 00:01:01,044 --> 00:01:02,546 SHE IS GOING TO TELL US ABOUT 19 00:01:02,546 --> 00:01:08,051 THE RESEARCH THERE AND I WANTED 20 00:01:08,051 --> 00:01:10,787 TO MENTION THAT THIS WAS MADE 21 00:01:10,787 --> 00:01:14,491 POSSIBLE BY ASMS GRANT FOR 22 00:01:14,491 --> 00:01:15,325 GENERAL DISCUSSION, MOST 23 00:01:15,325 --> 00:01:19,463 DISCUSSION GROUPS TO INVITE FOR 24 00:01:19,463 --> 00:01:22,466 THE DISCUSSION. 25 00:01:22,466 --> 00:01:23,367 THANK YOU. 26 00:01:23,367 --> 00:01:26,970 >> WOW, THANK YOU SO MUCH. 27 00:01:26,970 --> 00:01:29,506 SO BEFORE I DIG INTO THE SCIENCE 28 00:01:29,506 --> 00:01:33,176 AND TALK ABOUT PROXIMITY LABELS, 29 00:01:33,176 --> 00:01:35,245 PROTEOMICS AND GPR SIGNALING 30 00:01:35,245 --> 00:01:37,314 FIRST OF ALL I WANT TO THANK 31 00:01:37,314 --> 00:01:39,683 ALEX ANDROGENNIA AND JAN FOR 32 00:01:39,683 --> 00:01:40,984 HAVING ME HERE AND YOU FOR 33 00:01:40,984 --> 00:01:43,687 SHOWING UP AND GOING THROUGH THE 34 00:01:43,687 --> 00:01:45,022 COLD WEATHER. 35 00:01:45,022 --> 00:01:46,089 FROM CALIFORNIA COMING HERE, I 36 00:01:46,089 --> 00:01:46,890 WAS WORRIED ABOUT THE 37 00:01:46,890 --> 00:01:48,125 TEMPERATURE, I HAVE SEVERAL 38 00:01:48,125 --> 00:01:49,426 LAYERS BUT SO FAR I MANAGED 39 00:01:49,426 --> 00:01:49,760 QUITE WELL. 40 00:01:49,760 --> 00:01:53,797 AND WHAT I WANT TO MENTION IS 41 00:01:53,797 --> 00:01:56,133 ALSO IS IT'S A PLEASURE TO BE AT 42 00:01:56,133 --> 00:01:57,968 NIH AND IT'S ALMOST EXACTLY 20 43 00:01:57,968 --> 00:02:02,706 YEARS AGO THAT I WAS HERE WITH 44 00:02:02,706 --> 00:02:04,274 MY FIRST PROTEOMIC EXPERIMENT 45 00:02:04,274 --> 00:02:06,610 AND I ACTUALLY SOME SOME PHOTOS 46 00:02:06,610 --> 00:02:11,848 FROM A DIFFERENT TIME. 47 00:02:11,848 --> 00:02:15,018 SO THAT WAS HERE, 20 YEARS AGO 48 00:02:15,018 --> 00:02:17,254 IN THE BUILDING, THAT'S ME, YES, 49 00:02:17,254 --> 00:02:19,189 20 YEARS, SO THERE ARE SOME 50 00:02:19,189 --> 00:02:20,290 CHANGES THAT HAPPEN IN 20 YEARS 51 00:02:20,290 --> 00:02:21,592 AND IT WAS IN THE BUILDING 52 00:02:21,592 --> 00:02:23,493 ACROSS THE STREET AND THE 53 00:02:23,493 --> 00:02:25,596 BASEMENT AT THE TIME SONIA WAS 54 00:02:25,596 --> 00:02:28,298 LEADING THE MASS SPEC. FACILITY 55 00:02:28,298 --> 00:02:30,634 THERE SO I WAS THERE DURING 3 56 00:02:30,634 --> 00:02:32,603 MONTHS DURING MY MASTERS PROJECT 57 00:02:32,603 --> 00:02:34,571 AND THAT'S WHERE I STARTED MY 58 00:02:34,571 --> 00:02:35,706 PASSION ABOUT PROTEOMICS AND 59 00:02:35,706 --> 00:02:37,674 THAT WAS 20 YEARS AGO AND NEVER 60 00:02:37,674 --> 00:02:40,544 STOPPED AND STILL CONTINUES. 61 00:02:40,544 --> 00:02:42,379 TECHNOLOGY CHANGES, I HAVE 62 00:02:42,379 --> 00:02:49,252 CHANGED, AND FOR ME, --I DON'T 63 00:02:49,252 --> 00:02:50,020 KNOW ABOUT TECHNOLOGY. 64 00:02:50,020 --> 00:02:51,388 LET'S TALK ABOUT THE SCIENCE. 65 00:02:51,388 --> 00:02:54,524 IN GENERAL, I'M INTERESTED IN 66 00:02:54,524 --> 00:02:56,493 CELLS AND I SEE THE CELL AS A 67 00:02:56,493 --> 00:02:58,395 KIND OF COMPUTER WHICH RECEIVES 68 00:02:58,395 --> 00:03:02,032 A LOT OF SIGNALS FROM THE EXTRA 69 00:03:02,032 --> 00:03:03,667 CELLULAR ENVIRONMENT AND THEN 70 00:03:03,667 --> 00:03:04,968 SENSES THEM THROUGH RECEPTORS ON 71 00:03:04,968 --> 00:03:07,237 THE CELL SURFACE AND THEN THIS 72 00:03:07,237 --> 00:03:14,211 INFORMATION IS ON INSIDE THE 73 00:03:14,211 --> 00:03:15,379 CELL THROUGH BIOCHEMICAL 74 00:03:15,379 --> 00:03:16,813 INTERACTIONS THAT ARE MEDIATED 75 00:03:16,813 --> 00:03:21,685 BY THE PROTEINS AND AT THE END 76 00:03:21,685 --> 00:03:23,286 THERE'S CELLULAR RECEPTOR AS A 77 00:03:23,286 --> 00:03:24,688 RESPONSE OF THE CELL. 78 00:03:24,688 --> 00:03:28,659 SO OBVIOUSLY THIS IS NOT LIKE AN 79 00:03:28,659 --> 00:03:30,861 ON/OFF SWITCH, IT IS A 80 00:03:30,861 --> 00:03:32,129 COMPUTATION IN ITSELF AND RATHER 81 00:03:32,129 --> 00:03:34,164 IT'S HAPPENING IN THE SIGNALING 82 00:03:34,164 --> 00:03:35,632 NETWORKS AND IT'S A PROTEIN BUT 83 00:03:35,632 --> 00:03:40,537 I WOULD ARGUE THAT THIS IS 84 00:03:40,537 --> 00:03:41,238 DEFINED BY PROTEIN-PROTEIN 85 00:03:41,238 --> 00:03:43,407 INTERACTION AND HOW THEY ARE 86 00:03:43,407 --> 00:03:46,143 ORGANIZED IN TIME IS SPACE. 87 00:03:46,143 --> 00:03:47,411 SO NOW I'M SPECIFICALLY 88 00:03:47,411 --> 00:03:50,647 INTERESTED IN 1 CLASS OF 89 00:03:50,647 --> 00:03:52,349 G-PROTEIN RECEPTORS AND JUST TO 90 00:03:52,349 --> 00:03:55,052 GIVE YOU A PITCH WHY I THINK 91 00:03:55,052 --> 00:03:57,254 THESE ARE INTERESTING AND 92 00:03:57,254 --> 00:03:58,155 PROBABLY RPGHTS CAN AGREE TO 93 00:03:58,155 --> 00:04:00,624 THAT IS ON THE 1 SIDE THEY 94 00:04:00,624 --> 00:04:02,959 REPRESENT THE LARGEST FAMILY OF 95 00:04:02,959 --> 00:04:04,594 SIGNALING RECEPTORS, SO OVER 800 96 00:04:04,594 --> 00:04:05,896 OF THEM DIVERSE RECEPTORS ARE 97 00:04:05,896 --> 00:04:08,598 EXPRESSED IN OUR BODIES, ALL 98 00:04:08,598 --> 00:04:10,067 OVER, SO PHYSIOLOGICALLY THEY 99 00:04:10,067 --> 00:04:11,435 ARE HIGHLY RELEVANT BUT ON THE 100 00:04:11,435 --> 00:04:13,070 OTHER HAND THERE IS ALSO THE 101 00:04:13,070 --> 00:04:14,838 BIGGEST CLASS OF DRUG TARGETS SO 102 00:04:14,838 --> 00:04:16,940 OVER ONE-THIRD OF THE CURRENTLY 103 00:04:16,940 --> 00:04:23,413 APPROVED DRUGS, WE SEE HERE ARE 104 00:04:23,413 --> 00:04:24,648 TARGETING GPCR, BUT WHEN YOU 105 00:04:24,648 --> 00:04:26,416 LOOK AT THEM IN CLINICAL TRIALS 106 00:04:26,416 --> 00:04:31,288 AS SHOWN HERE FOR VARIOUS 107 00:04:31,288 --> 00:04:32,889 INDICATIONS, GPC Rs ARE THE 108 00:04:32,889 --> 00:04:33,523 DRUG TARGETS. 109 00:04:33,523 --> 00:04:35,692 SO UNDERSTANDING HOW THEY WORK 110 00:04:35,692 --> 00:04:42,265 IS QUITE IMPORTANT 111 00:04:42,265 --> 00:04:42,632 PHYSIOLOGICALLY. 112 00:04:42,632 --> 00:04:43,700 SO A BIG QUESTION WE ARE ASKING 113 00:04:43,700 --> 00:04:46,336 IN THE LAB IS HOW ARE WE 114 00:04:46,336 --> 00:04:47,304 TRACKING THE CELL ACROSS TIME 115 00:04:47,304 --> 00:04:47,771 AND SPACE. 116 00:04:47,771 --> 00:04:49,973 WHY DO WE DO THAT IN IF WE THINK 117 00:04:49,973 --> 00:04:52,542 ABOUT A BIG CLASS OF RECEPTORS 118 00:04:52,542 --> 00:04:54,544 THEY RESPOND TO DIFFERENT 119 00:04:54,544 --> 00:04:56,813 CHEMICALS, COMPOUNDS AND THERE 120 00:04:56,813 --> 00:05:00,217 ARE SOME EXAMPLES, IT'S LIKE A 121 00:05:00,217 --> 00:05:01,651 WIDE PALLET OF DIFFERENT 122 00:05:01,651 --> 00:05:03,954 SIGNALS, AND THEN THERE'S VERY 123 00:05:03,954 --> 00:05:06,990 DIVERSE CELLULAR AND 124 00:05:06,990 --> 00:05:08,692 PHYSIOLOGICAL RESPONSES THAT ARE 125 00:05:08,692 --> 00:05:10,127 KIND OF ARCH CHESTERATED THROUGH 126 00:05:10,127 --> 00:05:11,328 THE ACTIVATION OF RECEPTORS, SO 127 00:05:11,328 --> 00:05:13,063 THE QUESTION IS HOW DOES THE 128 00:05:13,063 --> 00:05:17,033 CELL COMPUTE ALL OF THIS, YEAH, 129 00:05:17,033 --> 00:05:17,934 INSIDE THE CELL. 130 00:05:17,934 --> 00:05:20,804 SO WHAT DO WE KNOW ABOUT GPCR 131 00:05:20,804 --> 00:05:24,674 RIGHT AND IT'S A SIMPLIFIED 132 00:05:24,674 --> 00:05:25,008 DIAGRAM HERE. 133 00:05:25,008 --> 00:05:27,177 SO FIRST WHEN YOU ACTIVATE THESE 134 00:05:27,177 --> 00:05:30,614 RECEPTORS, THEY ENGAGE THE 135 00:05:30,614 --> 00:05:31,414 HETEROTRI MERRIC G-PROTEIN AND 136 00:05:31,414 --> 00:05:38,155 IT CAN BE DIFFERENT FAMILIES OF 137 00:05:38,155 --> 00:05:40,357 THIS SYSTEM, GL5, THEY 138 00:05:40,357 --> 00:05:43,193 ACTIVATION DOWN STREAM PROCESSES 139 00:05:43,193 --> 00:05:44,694 AND MODULATE SECOND RESPONSES SO 140 00:05:44,694 --> 00:05:46,696 THE SIGNALING, THE G-PROTEIN 141 00:05:46,696 --> 00:05:47,864 SIGNALING IS TERMINATED BY 142 00:05:47,864 --> 00:05:50,700 PHOSPHORYLATION OF THE RECEPTORS 143 00:05:50,700 --> 00:05:52,502 THROUGH GRK, SO G-PROTEIN 144 00:05:52,502 --> 00:05:54,538 RECEPTOR KINASES WHICH IS THEN 145 00:05:54,538 --> 00:05:57,541 THOUGHT TO RECRUIT PROTEINS 146 00:05:57,541 --> 00:06:00,377 CALLED BETA ARRESTED THAT 147 00:06:00,377 --> 00:06:02,045 INITIATE INTERNALIZATION TO THE 148 00:06:02,045 --> 00:06:04,748 RECEPTOR TRAFFICKING TO THE 149 00:06:04,748 --> 00:06:06,550 COMPARTMENT SUCH AS ENDOSOMES, 150 00:06:06,550 --> 00:06:09,786 WHERE THEY SIGNAL TO THE 151 00:06:09,786 --> 00:06:13,490 COMPORTMENT AND THEN SIGNAL BACK 152 00:06:13,490 --> 00:06:14,691 TO THE BRAIN, AND IF YOU THINK 153 00:06:14,691 --> 00:06:17,360 ABOUT IT, THEY CAN BE MULTIPLE 154 00:06:17,360 --> 00:06:19,329 GPCR AND EXPRESSED IN THE SAME 155 00:06:19,329 --> 00:06:21,998 CELL, THEY ARE KNOWN TO GI, BUT 156 00:06:21,998 --> 00:06:23,934 TOWARD ANY OF THE PROTEIN, BUT 157 00:06:23,934 --> 00:06:25,836 THEY DO TOTALLY DIFFERENT 158 00:06:25,836 --> 00:06:26,937 THINGS, RIGHT? 159 00:06:26,937 --> 00:06:28,038 THE CELLS RESPOND DIFFERENTLY TO 160 00:06:28,038 --> 00:06:29,172 THE ACTIVATION SO WHEN YOU THINK 161 00:06:29,172 --> 00:06:31,007 ABOUT THIS, IT BECOMES CLEAR 162 00:06:31,007 --> 00:06:32,876 THERE MUST BE MORE 163 00:06:32,876 --> 00:06:33,510 SPECIALIZATION, ORGANIZATION ON 164 00:06:33,510 --> 00:06:36,012 A CELLULAR LEVEL THAT WE 165 00:06:36,012 --> 00:06:36,713 CURRENTLY DON'T UNDERSTAND. 166 00:06:36,713 --> 00:06:41,351 SO THAT'S THE VISION FOR MY LAB, 167 00:06:41,351 --> 00:06:44,221 BASICALLY IS TO UNCOVER THE 168 00:06:44,221 --> 00:06:46,089 BIOLOGICAL MECHANISM BEHIND THIS 169 00:06:46,089 --> 00:06:48,992 DIVERSE ORGANISM AND RESPONSE, 170 00:06:48,992 --> 00:06:50,293 SCIENCE IS A BIG GOAL BUT WHEN 171 00:06:50,293 --> 00:06:54,731 YOU START A LAB, YOU NEED BIG 172 00:06:54,731 --> 00:06:55,799 EXPERIMENTS AND FINDING SOME 173 00:06:55,799 --> 00:06:56,032 INTRS. 174 00:06:56,032 --> 00:06:59,536 SO IF WE HAVE DIFFERENT 175 00:06:59,536 --> 00:07:01,304 RECEPTORS THAT ARE ACTIVATED OR 176 00:07:01,304 --> 00:07:02,806 DIFFERENT RECEPTORS THAT ARE 177 00:07:02,806 --> 00:07:04,341 ACTIVATED WITH LIGAND THEY CAN 178 00:07:04,341 --> 00:07:06,610 LEAD TO DIFFERENT IN VIVO AND 179 00:07:06,610 --> 00:07:09,379 PHENOTYPES AND HOW IS THIS 180 00:07:09,379 --> 00:07:09,646 POSSIBLE? 181 00:07:09,646 --> 00:07:11,147 SO WE WANT TO FILL THIS SPACE 182 00:07:11,147 --> 00:07:12,682 AND THAT'S MY IDEA TO FILL THIS 183 00:07:12,682 --> 00:07:15,452 SPACE WITH MORE, TRY TO REALLY 184 00:07:15,452 --> 00:07:16,386 HAVE A MULTIDIMENSIONAL 185 00:07:16,386 --> 00:07:17,220 CHARACTERIZATION OF THE 186 00:07:17,220 --> 00:07:18,421 SIGNALING NETWORK THAT ARE 187 00:07:18,421 --> 00:07:21,524 ACTIVATED BY THE RECEPTORS BASED 188 00:07:21,524 --> 00:07:23,627 ON, YOU KNOW THE INTERACTIONS 189 00:07:23,627 --> 00:07:26,396 ABOUT THE REFORMS ABOUT, 190 00:07:26,396 --> 00:07:27,764 ACTIVATIONOT SIGNALING THAT ARE 191 00:07:27,764 --> 00:07:29,332 INITIATED AND WHERE THIS IS 192 00:07:29,332 --> 00:07:30,500 HAPPENING AND HOPEFULLY, RIGHT? 193 00:07:30,500 --> 00:07:32,936 THIS IS VERY SIMPLIFIED, THE 194 00:07:32,936 --> 00:07:33,870 EXPERIMENTS DATA NEVER LOOKED 195 00:07:33,870 --> 00:07:38,441 THAT GOOD BUT THE IDEA CAN BE 196 00:07:38,441 --> 00:07:39,743 THEN CORRELATED MOLECULAR EVENT 197 00:07:39,743 --> 00:07:41,778 WITH THE PHENOTYPES THAT WE CAN 198 00:07:41,778 --> 00:07:44,614 READ OUT AND THEN ON THE 1 HAND 199 00:07:44,614 --> 00:07:46,716 GET SOME IDEA ABOUT BIOLOGICAL 200 00:07:46,716 --> 00:07:48,418 MECHANISM, BUT OBVIOUSLY IN THE 201 00:07:48,418 --> 00:07:52,255 LONG RUN THIS HOPEFULLY INFORMED 202 00:07:52,255 --> 00:07:52,822 OF GPCR SIGNAL. 203 00:07:52,822 --> 00:07:55,258 SO THIS IS THE BIG VISION I WORK 204 00:07:55,258 --> 00:07:57,093 ON FOR MANY YEARS BUT WE HAVE 205 00:07:57,093 --> 00:07:59,429 MADE SMALL STEPS TOWARDS IT, AND 206 00:07:59,429 --> 00:08:01,731 THE FIRST THING WE NEED TO HAVE 207 00:08:01,731 --> 00:08:04,034 IS A METHOD THAT CAN TRACK THESE 208 00:08:04,034 --> 00:08:07,671 INTERACTIONS ON SIGNALING EVENTS 209 00:08:07,671 --> 00:08:09,406 THAT ARE ACTIVATED BY THE 210 00:08:09,406 --> 00:08:09,873 RECEPTORS. 211 00:08:09,873 --> 00:08:12,409 SO MY POST DOC SEVERAL YEARS 212 00:08:12,409 --> 00:08:14,377 BACK, WE DEVELOPED A METHOD 213 00:08:14,377 --> 00:08:17,247 WHERE WE USED AN ENZYME CALLED 214 00:08:17,247 --> 00:08:20,317 APEX, THE LABELING ENZYME AND 215 00:08:20,317 --> 00:08:21,484 ENGINEERED PEROXIDASE WHICH WE 216 00:08:21,484 --> 00:08:23,019 FUSE TO THE RECEPTOR. 217 00:08:23,019 --> 00:08:24,187 SOLET ADVANTAGE OF APEX IS WHEN 218 00:08:24,187 --> 00:08:26,289 YOU TREAT THE CELLS WITH BIOTIN 219 00:08:26,289 --> 00:08:29,926 ISSUES AND TO GIVE A VERY SHORT 220 00:08:29,926 --> 00:08:33,296 PAUSE OF HYDROGEN PEROXIDE AND 221 00:08:33,296 --> 00:08:37,267 THEN BIOTIN CAN ALWAYS TURN INTO 222 00:08:37,267 --> 00:08:39,002 RADICAL SAYS, IT'S HIGHLY 223 00:08:39,002 --> 00:08:41,738 REACTIVE AND IT'S LABELED IF A 224 00:08:41,738 --> 00:08:45,942 BIOTIN IS LAID PROTEIN IS IN THE 225 00:08:45,942 --> 00:08:47,544 PROXIMITY OF THE RECEPTOR, SO 226 00:08:47,544 --> 00:08:49,312 THIS IS NICE BECAUSE YOU HAVE 227 00:08:49,312 --> 00:08:51,748 LABELED PROTEINS NEXT TO THE 228 00:08:51,748 --> 00:08:52,315 RECEPTOR. 229 00:08:52,315 --> 00:08:53,249 THIS IS THE PROCESS TREATMENT, 230 00:08:53,249 --> 00:08:55,352 CAN YOU DO THIS BETWEEN 231 00:08:55,352 --> 00:08:57,854 30-SECONDS AND 1 MINUTE OR YOU 232 00:08:57,854 --> 00:09:00,223 CAN TAKE REALLY FOR PROTEOMICS, 233 00:09:00,223 --> 00:09:02,726 YOU CAN HAVE A HIGH TEMPORAL 234 00:09:02,726 --> 00:09:04,227 RESOLUTION SO IT TAKES A 235 00:09:04,227 --> 00:09:07,230 SNAPSHOT OF WHAT'S HAPPENING IN 236 00:09:07,230 --> 00:09:17,774 THE PROXIMITY OF THE RECEPTORS. 237 00:09:19,275 --> 00:09:20,810 NO PROBLEM. 238 00:09:20,810 --> 00:09:21,111 OKAY. 239 00:09:21,111 --> 00:09:26,182 NOW WE CAN CONTINUE. 240 00:09:26,182 --> 00:09:28,251 AND SO THE ADVANTAGE IS NOW WHEN 241 00:09:28,251 --> 00:09:30,086 YOU TAKE THESE SNAPSHOTS OVER 242 00:09:30,086 --> 00:09:31,321 THE TIME COURSE OF THE 243 00:09:31,321 --> 00:09:34,858 ACTIVATION, CAN YOU GET THESE 244 00:09:34,858 --> 00:09:36,126 PROXIMATE PROTEIN NETWORKS OF 245 00:09:36,126 --> 00:09:37,560 THE RECEPTOR OVER TIME, SO THEN 246 00:09:37,560 --> 00:09:39,429 THE NEXT STEP IS THEN, THIS 247 00:09:39,429 --> 00:09:40,597 HAPPENS WITHIN THE CELL, RIGHT, 248 00:09:40,597 --> 00:09:42,332 IN THE LIVING CELL, THEN, 249 00:09:42,332 --> 00:09:44,901 BECAUSE HAVE YOU MARKED THESE 250 00:09:44,901 --> 00:09:48,038 PROTEINS IN PROXIMITY, YOU CAN 251 00:09:48,038 --> 00:09:50,774 LICENSE THESE CELLS FOR ENRICH 252 00:09:50,774 --> 00:09:52,375 THEM WITH BIOTIN PROTEINS AND 253 00:09:52,375 --> 00:09:53,910 ENRICH THEM WITH PROTEOMICS AND 254 00:09:53,910 --> 00:09:55,111 WHAT WE'RE SHOWING IF WE USE 255 00:09:55,111 --> 00:09:56,880 THIS,A PROACH IS THAT WE CAN -- 256 00:09:56,880 --> 00:09:58,548 IT'S ACTUALLY THESE ARE KIND OF 257 00:09:58,548 --> 00:09:59,816 RICH DATA SETS, RIGHT? 258 00:09:59,816 --> 00:10:02,585 BECAUSE HERE LIBLED WITHIN THE 259 00:10:02,585 --> 00:10:03,653 RADIUS OF 10-20-NANOMETERS OR A 260 00:10:03,653 --> 00:10:05,555 LOT OF -- IN THE CELL, IT'S 261 00:10:05,555 --> 00:10:06,756 CROWDED, A LOT OF THINGS ARE 262 00:10:06,756 --> 00:10:08,925 HAPPENING BUT WHAT WE HAVE SHOWN 263 00:10:08,925 --> 00:10:10,860 IS THAT WE CAN CONVOLUNTEERSUDE 264 00:10:10,860 --> 00:10:11,928 THIS DATA SET AND GET 265 00:10:11,928 --> 00:10:13,229 INFORMATION ABOUT THE LOCATION 266 00:10:13,229 --> 00:10:17,467 OF THE RECEPTOR BY LABELING 267 00:10:17,467 --> 00:10:18,234 LOCATION SPECIFIC MARKERS OVER 268 00:10:18,234 --> 00:10:20,236 THE TIME POINT BUT ALSO THE TIME 269 00:10:20,236 --> 00:10:22,872 RESULT NETWORKS THAT ARE IN 270 00:10:22,872 --> 00:10:24,441 PROXIMITY THAT MIGHT FUNCTION 271 00:10:24,441 --> 00:10:26,876 WITH THE RECEPTOR. 272 00:10:26,876 --> 00:10:28,078 SO WE HAVE PUBLISHED THIS A 273 00:10:28,078 --> 00:10:30,113 WHILE AGO AND WE USED IT SINCE 274 00:10:30,113 --> 00:10:32,615 AND WE THOUGHT IT'S A QUITE 275 00:10:32,615 --> 00:10:33,917 POWERFUL METHOD TO IN AN 276 00:10:33,917 --> 00:10:35,752 UNBIASED WAY TO STUDY THESE 277 00:10:35,752 --> 00:10:36,052 NETWORKS. 278 00:10:36,052 --> 00:10:38,455 SO WHAT WAS THE LIMITATION, ONCE 279 00:10:38,455 --> 00:10:40,323 WE HAD IT WAS LIKE NO WE WANT TO 280 00:10:40,323 --> 00:10:42,792 APPLY THIS TO MANY LIGANDS AND 281 00:10:42,792 --> 00:10:43,760 ACTIVATE THE RECEPTORS, MANY 282 00:10:43,760 --> 00:10:44,594 DIFFERENT RECEPTORS BUT THEN YOU 283 00:10:44,594 --> 00:10:46,329 RUN INTO A PROBLEM, LIKE THAT'S 284 00:10:46,329 --> 00:10:47,864 ACTUALLY QUITE SOME WORK TO 285 00:10:47,864 --> 00:10:49,032 ENRICH THESE PROTEINS AND WE 286 00:10:49,032 --> 00:10:50,633 WANTED TO AUTOMATE IT. 287 00:10:50,633 --> 00:10:52,869 AND SO THIS IS WHAT WE HAVE BEEN 288 00:10:52,869 --> 00:10:55,338 PUT IN PLACE, RIGHT? 289 00:10:55,338 --> 00:10:57,307 IT'S ACTUALLY NOT ROCKET 290 00:10:57,307 --> 00:10:59,375 SCIENCE, BUT WE THOUGHT IT'S 291 00:10:59,375 --> 00:11:04,747 REALLY HELPFUL, IS THAT WE -- 292 00:11:04,747 --> 00:11:06,716 THE TIME LIMITING STUFF AT THAT 293 00:11:06,716 --> 00:11:07,784 TIME WAS THE ENRICHMENT, YOU 294 00:11:07,784 --> 00:11:09,986 HAVE TO DO A LOT OF WASHING 295 00:11:09,986 --> 00:11:11,488 STUFF, EVERYBODY THAT HAS 296 00:11:11,488 --> 00:11:13,857 PROTEIN ENRICHMENT IS AT THAT 297 00:11:13,857 --> 00:11:14,891 POINT QUITE TIME CONSUMING AND 298 00:11:14,891 --> 00:11:17,327 YOU CAN ONLY DO SO MANY SAMPLES 299 00:11:17,327 --> 00:11:18,962 AND WE HAVE AUTOMATED THIS 300 00:11:18,962 --> 00:11:21,164 PROCESS NOW ON A KINGFISHER 301 00:11:21,164 --> 00:11:22,065 FLEX. 302 00:11:22,065 --> 00:11:27,337 IT'S BASICALLY HAS 96 MAGNETS, 303 00:11:27,337 --> 00:11:29,105 MAGNETIC RODS AND HOW WE CAN DO 304 00:11:29,105 --> 00:11:31,608 THIS ENRICHMENT PROCESS AND 305 00:11:31,608 --> 00:11:33,676 WASHING STEP ALL AUTOMATED. 306 00:11:33,676 --> 00:11:38,248 THIS HAS BEEN REALLY NICE AND I 307 00:11:38,248 --> 00:11:41,151 SHOW YOU THIS, IT'S LIKE YOU CAN 308 00:11:41,151 --> 00:11:43,653 REALLY PLAY, IT'S LIKE NIX 309 00:11:43,653 --> 00:11:45,221 PLATES ON THE KING FISH CASE 310 00:11:45,221 --> 00:11:49,526 AUTOMATICALLY UNTIL WE DO THE 311 00:11:49,526 --> 00:11:51,027 BINDING OF THE BIOTINALATED 312 00:11:51,027 --> 00:11:51,895 PROTEINS AND WASH IT OUT THERE 313 00:11:51,895 --> 00:11:55,231 AND THEN WE HAVE THESE READY FOR 314 00:11:55,231 --> 00:11:55,498 SOLUTION. 315 00:11:55,498 --> 00:11:57,433 SO WHAT HELPS IS TO GET A HIGHER 316 00:11:57,433 --> 00:11:58,902 THROUGH PUT RIGHT BECAUSE WE CAN 317 00:11:58,902 --> 00:12:01,070 DO 96 AT THE SAME TIME AND THEN 318 00:12:01,070 --> 00:12:05,542 WE GET LOWER VARIABILITY BUT WE 319 00:12:05,542 --> 00:12:08,845 ALSO USE LOWER INPUT, WHICH ARE 320 00:12:08,845 --> 00:12:09,512 HARD TO DEMAND. 321 00:12:09,512 --> 00:12:13,449 SO JUST TO GIVE A BRIEF LIKE A 322 00:12:13,449 --> 00:12:15,919 LITTLE BIT ABOUT THE TECHNICAL 323 00:12:15,919 --> 00:12:17,420 PERFORMANCE IF YOU COMPARE THE 324 00:12:17,420 --> 00:12:19,155 MANUEL AND AUTOPLATE IT. 325 00:12:19,155 --> 00:12:21,691 IT'S LIKE INVASIVE PROTEIN 326 00:12:21,691 --> 00:12:22,625 IDENTIFICATION, WE SIMPLY 327 00:12:22,625 --> 00:12:24,561 COMPARE THE REPLICATE FROM THE 328 00:12:24,561 --> 00:12:27,564 SAME SAMPLE HERE, AND IT TERMS 329 00:12:27,564 --> 00:12:29,065 OF IDENTIFICATION, RIGHT, THE 330 00:12:29,065 --> 00:12:29,933 MASS SPEC PERFORMS PRETTY 331 00:12:29,933 --> 00:12:30,900 WELL, THE DIFFERENCES ARE NOT 332 00:12:30,900 --> 00:12:32,068 BETTER BUT NOT THAT MUCH, BUT 333 00:12:32,068 --> 00:12:34,304 THEN WHEN YOU LOOK AT HERE THE 334 00:12:34,304 --> 00:12:35,572 QUANTIFICATION, THE CO EFFICIENT 335 00:12:35,572 --> 00:12:37,340 OF VARIATION THAT THEY PLOT HERE 336 00:12:37,340 --> 00:12:39,309 ACROSS ALL PROTEINS IS THAT WHEN 337 00:12:39,309 --> 00:12:41,811 WE AUTOMATED IT, RIGHT, THE CO 338 00:12:41,811 --> 00:12:44,547 EFFICIENT VARIATION IS MUCH 339 00:12:44,547 --> 00:12:44,814 LOWER. 340 00:12:44,814 --> 00:12:49,619 SO WITH THAT, WE CAN DO THIS IN 341 00:12:49,619 --> 00:12:53,723 A MUCH SHORTER TIME AND A HIGHER 342 00:12:53,723 --> 00:12:54,090 REPRODUCIBILITY. 343 00:12:54,090 --> 00:12:56,593 SO WE WANT TO OF COURSE GET 344 00:12:56,593 --> 00:12:56,859 APPROVAL. 345 00:12:56,859 --> 00:12:58,828 CONCEPT RIGHT THAT WE CAN USE 346 00:12:58,828 --> 00:13:01,431 THIS AUTOMATED APPROACH COMBINED 347 00:13:01,431 --> 00:13:02,465 WITH DATA INDEPENDENT 348 00:13:02,465 --> 00:13:03,399 ACQUISITION FOR EVERYBODY THAT 349 00:13:03,399 --> 00:13:04,901 DOES PROTEIN COMPLEX OHMICS, WE 350 00:13:04,901 --> 00:13:07,237 LIKE IT BECAUSE IT'S ALWAYS 351 00:13:07,237 --> 00:13:08,137 REPRODUCED IN THE QUANTIFICATION 352 00:13:08,137 --> 00:13:10,306 AND NOW WE WANT TO SHOW PROOF OF 353 00:13:10,306 --> 00:13:10,540 CONCEPT. 354 00:13:10,540 --> 00:13:12,175 CAN WE USE THIS TO MAP 355 00:13:12,175 --> 00:13:13,876 INTERACTION OF NETWORK OF GPC 356 00:13:13,876 --> 00:13:16,045 Rs AND 1 RECEPTOR THAT WE 357 00:13:16,045 --> 00:13:22,285 TARTED OFF WITH IS THE SEROTONIN 358 00:13:22,285 --> 00:13:23,419 T2A RECEPTOR AND YOU SEE AGAIN 359 00:13:23,419 --> 00:13:26,489 IN THE WORK FLOW, WE FUSE THE 360 00:13:26,489 --> 00:13:30,226 APEX ENZYME TO THE C-TERMINUS OF 361 00:13:30,226 --> 00:13:32,095 THE RECEPTOR IN THE CELL AND WE 362 00:13:32,095 --> 00:13:33,396 HAVE THE T-CELLS, RIGHT, WE CAN 363 00:13:33,396 --> 00:13:35,164 ARGUE IT'S NOT ALL THE RECEPTORS 364 00:13:35,164 --> 00:13:36,399 AROUND THAT CELL LINE BUT IT'S 365 00:13:36,399 --> 00:13:37,867 AT THE BEGENERATEDDING WHEN YOU 366 00:13:37,867 --> 00:13:40,370 ESTABLISH THE METHOD, IS 367 00:13:40,370 --> 00:13:41,871 STRAIGHT FORWARD TO USE AND SO 368 00:13:41,871 --> 00:13:44,774 WE THEN ACTIVATE THE RECEPTOR, 369 00:13:44,774 --> 00:13:46,709 PERFORM THE PROXIMITY LAYER AT 370 00:13:46,709 --> 00:13:47,777 DIFFERENT TIME POINTS OF OVER 371 00:13:47,777 --> 00:13:49,979 THE TIME COURSE OF 30 MINUTES 372 00:13:49,979 --> 00:13:52,048 AND WE THEN, INNOVATOR TIME 373 00:13:52,048 --> 00:13:55,585 POINTS WE INTEGRATE INTO THESE 374 00:13:55,585 --> 00:13:57,053 TIME COURSE STATISTICS. 375 00:13:57,053 --> 00:13:59,889 SO WHAT WE CAN SEE THEN, IF YOU 376 00:13:59,889 --> 00:14:05,228 MODEL THE TIME COURSE DATA, THEN 377 00:14:05,228 --> 00:14:07,630 WE -- ENCLUSTERRED THE CHANGES 378 00:14:07,630 --> 00:14:11,701 IN THE PROXIMITY OF THE 379 00:14:11,701 --> 00:14:13,202 RECEPTOR, WE CLUSTER THEM INTO 380 00:14:13,202 --> 00:14:16,939 THE TRANSIENT EVENT BECAUSE IT'S 381 00:14:16,939 --> 00:14:18,341 HAPPENED, THIS INTERACTION EARLY 382 00:14:18,341 --> 00:14:20,677 AFTER RECEPTOR ACTIVATION AND 383 00:14:20,677 --> 00:14:22,679 THE MORE STABLER LATER 384 00:14:22,679 --> 00:14:24,180 INTERACTIONS THAT COME UP AT 385 00:14:24,180 --> 00:14:26,683 LATER TIMES IN TERMS OF 386 00:14:26,683 --> 00:14:27,050 ACTIVATION. 387 00:14:27,050 --> 00:14:29,218 AND JUST TO ZOOM INTO THIS 388 00:14:29,218 --> 00:14:30,520 TRANSIENT CLUSTER HERE IN THE 389 00:14:30,520 --> 00:14:32,655 BEGINNING, I MAPPED ALL THE 390 00:14:32,655 --> 00:14:34,290 PROTEINS FOR THE 4 CHANGES ON 391 00:14:34,290 --> 00:14:35,525 THE STRING INTERACTION, NETWORK 392 00:14:35,525 --> 00:14:37,060 AND WE ARE QUITE SAFE TO SEE 393 00:14:37,060 --> 00:14:38,795 RIGHT THERE, THERE ARE A NUMBER 394 00:14:38,795 --> 00:14:42,465 OF -- YOU SEE THEM COLOR CODED, 395 00:14:42,465 --> 00:14:43,766 LOOKING AT THE MODEL CITIZEN 396 00:14:43,766 --> 00:14:45,168 WILL KIEWL FUNCTION, THE 1S THAT 397 00:14:45,168 --> 00:14:46,002 ARE INDICATE INDEED BLUE ARE 398 00:14:46,002 --> 00:14:48,838 THINGS THAT HAVE BEEN REPORTED 399 00:14:48,838 --> 00:14:51,374 WE KNOW IN CONNECTION WITH GPCR 400 00:14:51,374 --> 00:14:54,143 SIGNALING AND FOR EXAMPLE, HERE, 401 00:14:54,143 --> 00:14:55,912 WE SEE THE ARRESTIN WHICH WAS I 402 00:14:55,912 --> 00:14:58,715 TALKING BEFORE, AS WELL AS GPC 403 00:14:58,715 --> 00:15:00,550 R14 KINASES, SO THIS IS QUITE 404 00:15:00,550 --> 00:15:04,387 NICE TO SEE THAT WE CAN NOW USE 405 00:15:04,387 --> 00:15:06,689 THIS TO MAP THE INTERACTION OF 406 00:15:06,689 --> 00:15:07,323 THE GFCRs. 407 00:15:07,323 --> 00:15:09,792 SO AS A SUMMARY, RIGHT, WE HAVE 408 00:15:09,792 --> 00:15:12,662 NOW THIS GPCR APEX, PROXIMITY 409 00:15:12,662 --> 00:15:15,398 LABELING METHOD THAT,A LOWS US 410 00:15:15,398 --> 00:15:16,666 TO MAP GPCS TEMPORAL RESOLUTION 411 00:15:16,666 --> 00:15:18,701 AND WE CAN DO THIS ACROSS 412 00:15:18,701 --> 00:15:25,441 MULTIPLE CONDITIONS BECAUSE WE 413 00:15:25,441 --> 00:15:26,376 HAVE MULTIPLE MAPPING. 414 00:15:26,376 --> 00:15:27,643 SO THAT'S THE INPUT AND NOW I 415 00:15:27,643 --> 00:15:31,447 WANT TO TALK ABOUT 2 416 00:15:31,447 --> 00:15:31,781 APPLICATIONS. 417 00:15:31,781 --> 00:15:34,317 THE FIRST 1 IS PUBLISHED 418 00:15:34,317 --> 00:15:36,185 ALREADY, BUT THE SECOND -- SORRY 419 00:15:36,185 --> 00:15:38,588 -- MULTIPLE PIECES THAT ARE NOT 420 00:15:38,588 --> 00:15:41,357 PUBLISHED YET BUT I HOPE IT'S AN 421 00:15:41,357 --> 00:15:42,759 INTERESTING DIRECTION WHERE WE 422 00:15:42,759 --> 00:15:43,426 ARE HEADING NOW. 423 00:15:43,426 --> 00:15:46,362 SO IN THE FIRST PART I WANT TO 424 00:15:46,362 --> 00:15:48,431 TALK ABOUT WHERE WE USE THIS 425 00:15:48,431 --> 00:15:50,800 PROXIMITY LABELING METHOD TO 426 00:15:50,800 --> 00:15:57,540 STUDY THE EFFECTS ON THE 427 00:15:57,540 --> 00:15:58,374 MU-OPIOID RECEPTOR, AS MOST OF 428 00:15:58,374 --> 00:16:00,977 YOU KNOW ISSUES IT'S FOR PAIN 429 00:16:00,977 --> 00:16:03,279 MEDICATION AND BECAUSE OF THE 430 00:16:03,279 --> 00:16:08,418 OPIOID CRISIS, THERE IS A LOT OF 431 00:16:08,418 --> 00:16:10,586 STUDIES AND MEDICATIONS THAT ARE 432 00:16:10,586 --> 00:16:11,187 LESS ADDICTIVE AND SIDE CENTER 433 00:16:11,187 --> 00:16:12,221 FOR CENTER 434 00:16:12,221 --> 00:16:15,825 FOR EFFECTS 435 00:16:15,825 --> 00:16:16,759 AND THERE'S AN INTEREST IN 436 00:16:16,759 --> 00:16:18,628 TRYING TO UNDERSTAND IF YOU HAVE 437 00:16:18,628 --> 00:16:20,963 ANY DIFFERENT LIGAND FOR THE 438 00:16:20,963 --> 00:16:22,465 RECEPTOR, HAVE 1 OF THE 439 00:16:22,465 --> 00:16:25,701 DIFFERENT REGULATORY MECHANISMS 440 00:16:25,701 --> 00:16:27,670 AND SO THIS -- THIS IS MORE 441 00:16:27,670 --> 00:16:30,907 PROOF OF CONCEPT STUDY, THIS IS 442 00:16:30,907 --> 00:16:32,642 ALSO HAS IMPLICATIONS, AND IF IT 443 00:16:32,642 --> 00:16:34,410 CAN EXPAND THIS TO A LARGE 444 00:16:34,410 --> 00:16:35,211 NUMBER OF LIGANDS. 445 00:16:35,211 --> 00:16:37,079 SO NOW WE CHOSE HERE AS A 446 00:16:37,079 --> 00:16:40,483 STARTING -- AS A STARTING SAID, 447 00:16:40,483 --> 00:16:42,785 3 DIFFERENT LIGANDS THAT ARE 448 00:16:42,785 --> 00:16:48,024 CHEMICALLY QUITE DIVERSE, SO WE 449 00:16:48,024 --> 00:16:52,962 CHOSE ANOTHER LIGAND, AND WE 450 00:16:52,962 --> 00:16:55,164 CHOSE THE PZM21 IS A NEW LIGAND 451 00:16:55,164 --> 00:16:56,699 OF CHEMISTRY THAT WAS DEVELOPED 452 00:16:56,699 --> 00:16:58,367 A FEW YEARS BACK. 453 00:16:58,367 --> 00:17:00,002 AND SO WE'RE ASKING THE QUESTION 454 00:17:00,002 --> 00:17:03,940 IF WE USE THE PROXIMITY LABELING 455 00:17:03,940 --> 00:17:05,808 APPROACH OVER A TIME COURSE OF 456 00:17:05,808 --> 00:17:07,844 60 MINUTES HOW DIFFERENT DO 457 00:17:07,844 --> 00:17:10,913 THESE INTERACTION NETWORKS LOOK 458 00:17:10,913 --> 00:17:13,516 LIKE WITHOUT PROTEOMIC METHOD. 459 00:17:13,516 --> 00:17:16,419 AND ALSO, ON THE DATA RIGHT, I 460 00:17:16,419 --> 00:17:19,555 TOLD YOU BEFORE THAT WE -- WE 461 00:17:19,555 --> 00:17:22,992 CAN MAP THE DIFFERENCES IN THE 462 00:17:22,992 --> 00:17:25,061 -- SORRY, THAT WE CAN MAP 463 00:17:25,061 --> 00:17:26,128 DIFFERENCES IN THE CELLULAR 464 00:17:26,128 --> 00:17:27,430 LOCATION OF THE RECEPTOR FAITHS 465 00:17:27,430 --> 00:17:28,698 BAGS OFFICE OF DIVERSITY 466 00:17:28,698 --> 00:17:30,199 PROTEINS AND PROXIMITY BUT ALSO 467 00:17:30,199 --> 00:17:30,933 THE INTERACTION NETWORK AND I 468 00:17:30,933 --> 00:17:33,469 WANT TO SHOW YOU 2 PIECES OF THE 469 00:17:33,469 --> 00:17:35,104 DATA NOW, SUMMARIZE THE STUDY, 470 00:17:35,104 --> 00:17:36,072 SO THE WORN PIECE OF THE STUDY 471 00:17:36,072 --> 00:17:37,907 IS THAT I WANT TO SHOW YOU THE 472 00:17:37,907 --> 00:17:39,408 DIFFERENCE IN CELLULAR LOCATION 473 00:17:39,408 --> 00:17:41,477 THAT WILL PREDICT 1 FROM THE 474 00:17:41,477 --> 00:17:42,812 PROTEOMICS DATA AND THAT'S SHOWN 475 00:17:42,812 --> 00:17:49,185 HERE, SO WE HAVE HERE THE 476 00:17:49,185 --> 00:17:51,687 DIFFERENT CAUSE AND LOCATION, 477 00:17:51,687 --> 00:17:53,155 THEY INDICATE LOCATION ON THE 478 00:17:53,155 --> 00:17:54,056 RECEPTOR THAT WE PROTECT DICT ON 479 00:17:54,056 --> 00:17:55,725 THE MOTE I DON'T MEANIC DATA AND 480 00:17:55,725 --> 00:17:58,094 WHAT WE CAN SEE HERE AND I FIND 481 00:17:58,094 --> 00:17:59,629 REMARKABLE IS THAT BASED ON THE 482 00:17:59,629 --> 00:18:01,364 PROTEOMIC DATA WE CAN REALLY SEE 483 00:18:01,364 --> 00:18:02,465 HOW THE RECEPTOR MOVED THROUGH 484 00:18:02,465 --> 00:18:05,301 THE CELLS, SO WHEN THE RECEPTORS 485 00:18:05,301 --> 00:18:06,502 ACTIVATED WITH THEM, YOU SEE THE 486 00:18:06,502 --> 00:18:08,204 MAIORITY OF THE RECEPTOR, AT 487 00:18:08,204 --> 00:18:09,605 LEAST AS THE PLASMA MEMBRANE, 488 00:18:09,605 --> 00:18:11,741 AND THEN THE PLASMA MEMBRANE 489 00:18:11,741 --> 00:18:13,209 LOCATION DECREASES OVER TIME, 490 00:18:13,209 --> 00:18:16,779 THE RECEPTOR IS INTERNALIZED AND 491 00:18:16,779 --> 00:18:18,748 RELOCALLIZED AND PART OF IT TO 492 00:18:18,748 --> 00:18:20,149 THE LYSOSOME, NOW WE'RE 493 00:18:20,149 --> 00:18:20,983 ACTIVATED WITH MORPHINE, IT'S 494 00:18:20,983 --> 00:18:23,853 KNOWN TO BE A PARTIAL AGONIST, 495 00:18:23,853 --> 00:18:30,927 SO IT DOESN'T REACH THE SAME 496 00:18:30,927 --> 00:18:32,962 ACTIVITY THAN DAMGO, SO NOT AS 497 00:18:32,962 --> 00:18:37,166 MUCH OF THE RECEPTOR IS 498 00:18:37,166 --> 00:18:39,268 INTERNALIZED, AND WITH PZM21, IT 499 00:18:39,268 --> 00:18:40,703 LOOKS VERY DIFFERENT, THE 500 00:18:40,703 --> 00:18:42,104 RECEPTOR STAYSA THE PLASMA 501 00:18:42,104 --> 00:18:42,371 MEMBRANE. 502 00:18:42,371 --> 00:18:46,609 AND WE CAN GET THIS FROM THE 503 00:18:46,609 --> 00:18:47,543 PROTEOMIC DATA. 504 00:18:47,543 --> 00:18:49,745 BUT NOW THE QUESTION IS HOW IS 505 00:18:49,745 --> 00:18:51,213 THE DIFFERENCE IN THE 506 00:18:51,213 --> 00:18:51,681 LOCALIZATION? 507 00:18:51,681 --> 00:18:53,716 HOW IS THE DIFFERENCE IN THE 508 00:18:53,716 --> 00:18:54,917 INTERACTIVE NETWORKS THAT ARE 509 00:18:54,917 --> 00:18:55,985 ASSOCIATED BY THE RECEPTOR, 510 00:18:55,985 --> 00:18:56,185 RIGHT? 511 00:18:56,185 --> 00:18:57,887 AND I SHOW YOU HERE, I SHOW THIS 512 00:18:57,887 --> 00:19:00,222 HERE IN FORM OF THE HEAT MAP, SO 513 00:19:00,222 --> 00:19:01,924 WHAT YOU SEE, THE HEAT OR THE 514 00:19:01,924 --> 00:19:06,228 DIFFERENT CAUSE HERE IS THE 515 00:19:06,228 --> 00:19:08,064 CHANGE IN BIOTINNATION OVER THE 516 00:19:08,064 --> 00:19:11,167 TIME COURSE, THE MORE RED IT IS, 517 00:19:11,167 --> 00:19:11,867 THE STRONGER BIOTINNATION, HAVE 518 00:19:11,867 --> 00:19:15,104 YOU AND I HOPE YOU CAN 519 00:19:15,104 --> 00:19:16,706 APPRECIATE FROM THIS THAT NOT 520 00:19:16,706 --> 00:19:18,240 ONLY THAT'S A BIG DIFFERENCE IN 521 00:19:18,240 --> 00:19:19,775 LOCALIZATION OF THE RECEPTOR, 522 00:19:19,775 --> 00:19:21,477 BUT IT ALSO LOOKED QUITE 523 00:19:21,477 --> 00:19:22,445 DRAMATIC THE DIFFERENCE IN THE 524 00:19:22,445 --> 00:19:24,080 PROTEINS THAT ARE ENGAGED AND 525 00:19:24,080 --> 00:19:25,781 THAT MAKES PROBABLY SENSE RIGHT 526 00:19:25,781 --> 00:19:28,351 BECAUSE OF THAT DEFINITE 527 00:19:28,351 --> 00:19:29,251 TRAFFICKING TRAJECTORY OF THE 528 00:19:29,251 --> 00:19:30,686 RECEPTOR, SO WHAT WE SEE IS THAT 529 00:19:30,686 --> 00:19:32,622 THE BIGGEST EFFECT IN TERMS OF 530 00:19:32,622 --> 00:19:35,157 THE CHANGES IN THE PROTEOME IS 531 00:19:35,157 --> 00:19:37,393 WHEN THE RECEPTOR IS ACTIVATED 532 00:19:37,393 --> 00:19:38,561 WITHIN DAMGO AND THERE ARE 533 00:19:38,561 --> 00:19:43,165 LITTLE EFFECTS WE SEE WITH P21 534 00:19:43,165 --> 00:19:47,136 -- WHEN WE ACTIVATE THE RECEPTOR 535 00:19:47,136 --> 00:19:47,770 OF P21. 536 00:19:47,770 --> 00:19:50,573 WHEN WE PERFORM A SIMPLE GENE 537 00:19:50,573 --> 00:19:51,240 ONTOLOGY ENRICHMENT ANALYSIS TO 538 00:19:51,240 --> 00:19:52,108 UNDERSTAND WHAT MIGHT BE THE 539 00:19:52,108 --> 00:19:53,309 FUNCTION OF ALL THESE PROTEINS 540 00:19:53,309 --> 00:19:56,278 IS THAT WHEN WE ACTIVATE THE 541 00:19:56,278 --> 00:20:00,683 RECEPTOR WITH D AMGO, WE ENRICH 542 00:20:00,683 --> 00:20:03,386 THE PROTEINS THAT ARE MEDIATING 543 00:20:03,386 --> 00:20:04,353 TRANSPORT AND ENDOCYTOSIS, RIGHT 544 00:20:04,353 --> 00:20:05,755 AND THAT DEFINITE LOW MAKES 545 00:20:05,755 --> 00:20:08,858 SENSE AND THERE'S NONE OF THESE 546 00:20:08,858 --> 00:20:12,528 PROTEINS ARE THERE WHEN WE 547 00:20:12,528 --> 00:20:14,397 ACTIVATE THE RECEPTOR WITH P21. 548 00:20:14,397 --> 00:20:16,198 SO WE WENT AHEAD AND WE WANT TO 549 00:20:16,198 --> 00:20:17,600 SAY, WELL, IF WE HAVE THIS DATA 550 00:20:17,600 --> 00:20:20,770 HERE IT CAN BE VALIDATED SOME OF 551 00:20:20,770 --> 00:20:21,537 THESE PROTEINS, ESPECIALLY THE 552 00:20:21,537 --> 00:20:23,239 1S THAT ARE ASSOCIATED WITH 553 00:20:23,239 --> 00:20:26,242 THEM, ARE REALLY REGULATE 554 00:20:26,242 --> 00:20:27,510 RECEPTOR INTERNALIZATION AND 555 00:20:27,510 --> 00:20:34,750 TRAFFICKING, AND SO TO DO THAT, 556 00:20:34,750 --> 00:20:38,521 WE DESIGN -- WE -- OKAY, 557 00:20:38,521 --> 00:20:41,123 SOMEBODY'S NOT ON MUTE. 558 00:20:41,123 --> 00:20:43,893 SO WE DESEEN A FLOW CYTOMETRY 559 00:20:43,893 --> 00:20:46,328 BASED MEASURE TO MEASURE 560 00:20:46,328 --> 00:20:47,563 RECEPTOR TRAFFICKING BUT THAT'S 561 00:20:47,563 --> 00:20:50,199 DIFFICULT BECAUSE THEN WE CAN DO 562 00:20:50,199 --> 00:20:51,367 MULTIPLE PERTURBATIONS AND WE 563 00:20:51,367 --> 00:20:53,102 CAN SEE THE TRAJECTORY. 564 00:20:53,102 --> 00:20:58,708 AND TO DEVELOP THIS, WE WORKED 565 00:20:58,708 --> 00:21:02,078 TOGETHER WITH THE WILLOW COYOTO 566 00:21:02,078 --> 00:21:04,480 LAB AT UCSF, AND WE USED A HELLO 567 00:21:04,480 --> 00:21:07,016 TAG WHICH WE FUSE INTO THE END 568 00:21:07,016 --> 00:21:09,452 TERMINUS OF THE RECEPTOR, SO THE 569 00:21:09,452 --> 00:21:13,155 ADVANTAGE OF THE HALO TAG IS 570 00:21:13,155 --> 00:21:14,557 THAT IT CAN DEFINE DIFFERENT 571 00:21:14,557 --> 00:21:15,958 LIGAND ANDS THEY ARE FLUORESCENT 572 00:21:15,958 --> 00:21:19,261 SO THEY CAN BE FLUORESCENT AND 573 00:21:19,261 --> 00:21:20,429 THEN DIFFERENT CHEMISTRIES ABOUT 574 00:21:20,429 --> 00:21:23,599 THESE LIGANDS SO THEY EXIST IN 575 00:21:23,599 --> 00:21:25,701 CELLS PERMEABLE AND CELL 576 00:21:25,701 --> 00:21:26,268 IMPERMEABLE AND NATURE. 577 00:21:26,268 --> 00:21:29,105 SO WHAT IS NICE NOW IS THAT WE 578 00:21:29,105 --> 00:21:32,074 CAN USE THESE SEQUEPTIALLY. 579 00:21:32,074 --> 00:21:33,709 WE CAN FIRST SATURATE, WE CAN 580 00:21:33,709 --> 00:21:35,111 USE THOSE, AND SATURATE ALL THAT 581 00:21:35,111 --> 00:21:37,646 STUFF IN THE PLASMA MEMBRANE 582 00:21:37,646 --> 00:21:38,481 WITH 1 FLUORESCENT COLOR AND 583 00:21:38,481 --> 00:21:40,549 THEN WE CAN DO A SECOND DAY 584 00:21:40,549 --> 00:21:47,356 LABELING WHERE WE LABEL, WITH 585 00:21:47,356 --> 00:21:48,924 THE PERMEABLE DYE, WE LABEL THE 586 00:21:48,924 --> 00:21:49,859 RECEPTORS AND WE LABEL THEM 587 00:21:49,859 --> 00:21:51,260 INSIDE THE CELL WITH A DIFFERENT 588 00:21:51,260 --> 00:21:51,494 COLOR. 589 00:21:51,494 --> 00:21:54,029 SO THIS IS NEAT, AND WE CAN READ 590 00:21:54,029 --> 00:21:56,465 OUT BY FLOW SIGNIFY TEMETRY, 591 00:21:56,465 --> 00:21:58,768 BASED ON A FLUORESCENT READ OUT 592 00:21:58,768 --> 00:22:00,736 HOW MUCH WAS ON THE CELL SURFACE 593 00:22:00,736 --> 00:22:02,204 AND INSIDE CELL. 594 00:22:02,204 --> 00:22:04,507 AND TO SHOW THAT THERE'S WORK 595 00:22:04,507 --> 00:22:08,110 HERE, HERE'S AN IMAGE WHERE THE 596 00:22:08,110 --> 00:22:09,745 IMAGE FOR THE DIFFERENT CORNERS 597 00:22:09,745 --> 00:22:12,481 WE HAVE HERE, FOR THE RECEPTOR, 598 00:22:12,481 --> 00:22:15,251 AND IT'S AFTER ACTIVATION, SEE 599 00:22:15,251 --> 00:22:17,419 THERE'S' RECEPTOR IN THE CELL 600 00:22:17,419 --> 00:22:22,691 SURFACE, WHICH IS LABELED WITH 601 00:22:22,691 --> 00:22:24,693 THE OTHER WE SEE A SUBSTANTIAL 602 00:22:24,693 --> 00:22:25,528 AMOUNT OF RECEPTOR WITHIN THE 603 00:22:25,528 --> 00:22:29,465 CELL AND WHEN WE CO STAIN THIS 604 00:22:29,465 --> 00:22:31,267 WITH A MARKER THAT'S ALREADY IN 605 00:22:31,267 --> 00:22:31,901 THE ENDOSTUDIES OF MULTIPLE 606 00:22:31,901 --> 00:22:34,904 ENDOCRINE, WE SEE THERE'S A GOOD 607 00:22:34,904 --> 00:22:36,205 COLOCALIZATION, RIGHT? 608 00:22:36,205 --> 00:22:37,173 INDICATING THAT THE RECEPTOR 609 00:22:37,173 --> 00:22:40,509 INSIDE THE CELL IS AT THE 610 00:22:40,509 --> 00:22:40,943 ENDOSOME. 611 00:22:40,943 --> 00:22:42,812 SO THIS LABELED WORK, AND WE CAN 612 00:22:42,812 --> 00:22:44,213 LABEL THE CELL WITH THE 613 00:22:44,213 --> 00:22:46,515 DIFFERENT COLORS AND CAN READ 614 00:22:46,515 --> 00:22:48,017 THIS WITH ACTUALLY CYTOMETRY. 615 00:22:48,017 --> 00:22:49,652 SO WE THEN COMBINE THESE 616 00:22:49,652 --> 00:22:52,288 FLUORESCENT READ OUTS WITH KNOCK 617 00:22:52,288 --> 00:22:53,889 OUTS, AS WE FOUND IN THE 618 00:22:53,889 --> 00:22:55,524 INTERACTION OF THE WORK OF THE 619 00:22:55,524 --> 00:22:56,826 RECEPTOR, SO THIS IS HOW THE 620 00:22:56,826 --> 00:23:01,664 WORK FLOW LOOKS LIKE, WE HAVE 621 00:23:01,664 --> 00:23:03,966 OUR CELLS THAT EXPRESS THE CELL 622 00:23:03,966 --> 00:23:07,336 SET HERE, AND THEN WE KNOCK OUT 623 00:23:07,336 --> 00:23:08,070 THE SELECT CERTAIN FACTORS THAT 624 00:23:08,070 --> 00:23:10,539 I WILL SHOW YOU NOW, IF WE KNOCK 625 00:23:10,539 --> 00:23:12,842 THEM OUT, WE HAVE THE CELLS AND 626 00:23:12,842 --> 00:23:15,811 WE PERFORM THIS ASSAY, WITHIN 627 00:23:15,811 --> 00:23:16,812 AND WITHOUT RECEPTOR ACTIVATION 628 00:23:16,812 --> 00:23:18,347 AND WE CAN MEASURE THE 629 00:23:18,347 --> 00:23:22,218 DIFFERENCE IN CELL SURFACE 630 00:23:22,218 --> 00:23:24,720 RECEPTOR VERSUS INSIDE THE CELL. 631 00:23:24,720 --> 00:23:27,556 AND WE SELECTED FROM OUR 632 00:23:27,556 --> 00:23:29,725 INTERACTION NETWORK, WE SELECTED 633 00:23:29,725 --> 00:23:36,031 8 NETWORK COMPONENTS THAT ARE 634 00:23:36,031 --> 00:23:38,567 PREDOMINANTLY LABELED BIOTIN AND 635 00:23:38,567 --> 00:23:40,970 ACTIVATED WITH DAMGO, WE SHOWED 636 00:23:40,970 --> 00:23:43,439 ARRESTIN AS A POSITIVE CONTROL, 637 00:23:43,439 --> 00:23:44,273 THIS IS INTERNALIZATION, IF WE 638 00:23:44,273 --> 00:23:46,242 KNOCK THIS OUT WE WOULD EXPECT 639 00:23:46,242 --> 00:23:47,643 THERE'S LESS RECEPTOR AND 640 00:23:47,643 --> 00:23:49,378 INTERNALIZED AND MORE ON THE 641 00:23:49,378 --> 00:23:52,081 PLASMA MEMBRANE, OF COURSE WE 642 00:23:52,081 --> 00:23:52,715 INCLUDED NONTARGETTING CONTROLS. 643 00:23:52,715 --> 00:23:54,850 AND JUST TO SHOW YOU HOW THESE 644 00:23:54,850 --> 00:23:56,085 RESULTS LOOK LIKE, I WANT TO 645 00:23:56,085 --> 00:23:57,653 WALK THROUGH THIS HERE, SO 646 00:23:57,653 --> 00:24:01,056 HERE'S FIRST THE NONTARGETTING 647 00:24:01,056 --> 00:24:02,925 CONTROL, WHAT WE PLOT HERE IS 648 00:24:02,925 --> 00:24:04,093 THE NORMALIZED RATIO BETWEEN 649 00:24:04,093 --> 00:24:05,861 RECEPTOR AT THE CELL SURFACE AND 650 00:24:05,861 --> 00:24:06,862 INSIDE THE CELL. 651 00:24:06,862 --> 00:24:09,031 SO THIS IS THAT TIME POINT 652 00:24:09,031 --> 00:24:10,299 THEORIZE THAT WE NORMALIZE THIS 653 00:24:10,299 --> 00:24:12,201 HERE AND WHEN WE ACTIVATE THE 654 00:24:12,201 --> 00:24:13,802 RECEPTOR FOR THE NONTARGETTING 655 00:24:13,802 --> 00:24:16,472 CONTROL, WE SEE THAT SUBSTANTIAL 656 00:24:16,472 --> 00:24:18,207 AMOUNT OF RECEPTOR, THE RACE 657 00:24:18,207 --> 00:24:20,643 CHANGES BECAUSE OF SUBSTANTIAL 658 00:24:20,643 --> 00:24:21,677 RECEPTOR INTERNALIZES. 659 00:24:21,677 --> 00:24:23,879 NOW WHEN WE KNOCK OUT ARRESTIN, 660 00:24:23,879 --> 00:24:27,149 WE CAN SEE THERE'S MUCH LESS OF 661 00:24:27,149 --> 00:24:27,917 THE RECEPTORS INTERNALIZING AND 662 00:24:27,917 --> 00:24:30,286 WE SEE A LITTLE OF THIS BECAUSE 663 00:24:30,286 --> 00:24:31,787 IT'S FOLLICULAR AND KNOCK OUT, 664 00:24:31,787 --> 00:24:32,988 IT'S PROBABLY NOT A HUNDRED 665 00:24:32,988 --> 00:24:33,422 PERCENT KNOCK OUT. 666 00:24:33,422 --> 00:24:34,723 SO THIS IS QUITE NICE. 667 00:24:34,723 --> 00:24:36,025 IT'S A POSITIVE CONTROLS AND WE 668 00:24:36,025 --> 00:24:37,359 WERE WONDERING FOR THE OTHERS, 669 00:24:37,359 --> 00:24:42,464 AND WE CHOSE THAT, HOW THAT 670 00:24:42,464 --> 00:24:43,799 DISTRIBUTION, AND INTERNALIZED 671 00:24:43,799 --> 00:24:45,768 RECEPTOR CHANGES AND WHAT WE SAW 672 00:24:45,768 --> 00:24:48,671 IS THAT FOR 2 PROTEINS I 673 00:24:48,671 --> 00:24:53,309 HIGHLIGHT THEM, AND BPS 35, WE 674 00:24:53,309 --> 00:24:54,443 SEE A SIGNIFICANT DIFFERENCE BUT 675 00:24:54,443 --> 00:24:57,579 IT LOOKED VERY DIFFERENT TO 676 00:24:57,579 --> 00:24:58,080 ARRESTIN, RIGHT? 677 00:24:58,080 --> 00:25:05,854 SO BASED ON THIS DATA, WE 678 00:25:05,854 --> 00:25:08,590 HYPOTHESIZED NOW, THAT COMMD3 679 00:25:08,590 --> 00:25:10,259 AND IT'S MORE RECYCLING OF THE 680 00:25:10,259 --> 00:25:11,660 RECEPTOR BACK TO THE PLASMA 681 00:25:11,660 --> 00:25:14,830 MEMBRANE AND THAT'S WHERE WE SEE 682 00:25:14,830 --> 00:25:17,199 HERE ALREADY AT STEADY STATE 683 00:25:17,199 --> 00:25:22,638 WHERE WE HAVE SOME ALL RIGHT 684 00:25:22,638 --> 00:25:23,439 SOME ACTIVATED RECEPTOR. 685 00:25:23,439 --> 00:25:25,541 WE SEE THERE'S LESS RECEPTOR AT 686 00:25:25,541 --> 00:25:27,076 THE CELL SURFACE AND IN FACT, 687 00:25:27,076 --> 00:25:29,178 THERE'S A RECENT PAPER THAT CAME 688 00:25:29,178 --> 00:25:31,347 OUT THAT CHARACTERIZED MORE 689 00:25:31,347 --> 00:25:34,650 INTENSE 35, IS A PROTEIN PART OF 690 00:25:34,650 --> 00:25:36,719 THE COMPLEX THAT ACTUALLY SHOW 691 00:25:36,719 --> 00:25:39,221 THAT CONTEXT, INVOLVED IN 692 00:25:39,221 --> 00:25:41,190 RECYCLING OF THE NEW OPIOID 693 00:25:41,190 --> 00:25:42,358 RECEPTOR AND THAT MAKES SENSE 694 00:25:42,358 --> 00:25:43,592 WITH OUR FINDING WHICH IS ALWAYS 695 00:25:43,592 --> 00:25:44,126 GOOD TO SEE. 696 00:25:44,126 --> 00:25:47,396 SO JUST TO SUMMARIZE THIS, WE 697 00:25:47,396 --> 00:25:48,130 VALIDATED OTHER PROTEINS WE 698 00:25:48,130 --> 00:25:49,999 FOUND HERE THAT FUNCTION IN 699 00:25:49,999 --> 00:25:50,699 G-PROTEIN SIGNALING BUT I DON'T 700 00:25:50,699 --> 00:25:51,800 HAVE TIME TO GO THROUGH ALL OF 701 00:25:51,800 --> 00:25:52,968 THIS BECAUSE I WANT TO TELL YOU 702 00:25:52,968 --> 00:25:54,770 A BIT ABOUT SOMETHING ELSE, 703 00:25:54,770 --> 00:25:56,038 THAT'S NOT PUBLISHED YET, BUT 704 00:25:56,038 --> 00:25:57,806 JUST TO SUMMARIZE THIS PART, I 705 00:25:57,806 --> 00:26:00,676 THINK WE HAVE, YOU KNOW A 706 00:26:00,676 --> 00:26:02,878 POWERFUL UNBIASED APPROACH FOR 707 00:26:02,878 --> 00:26:04,380 CHARACTERIZING DISTINCT LIGANDS 708 00:26:04,380 --> 00:26:05,948 THAT TARGET GPC Rs AND ALLOWED 709 00:26:05,948 --> 00:26:08,484 US BASICALLY TO LOOKING ACROSS 710 00:26:08,484 --> 00:26:09,852 THE DIFFERENT LIGANDS ALLOWED US 711 00:26:09,852 --> 00:26:12,488 TO DISCOVER ANOTHER REGULATORS 712 00:26:12,488 --> 00:26:13,555 FOR SIGNALING AND TRAFFICKING 713 00:26:13,555 --> 00:26:15,958 AND SO SPECIFICALLY HERE, I 714 00:26:15,958 --> 00:26:18,927 SHOWED YOU THAT WE COULD MAP THE 715 00:26:18,927 --> 00:26:19,895 DIFFERENT TRAFFICKING BEHAVIOR 716 00:26:19,895 --> 00:26:21,864 OF THE RECEPTOR BASED ON THE 717 00:26:21,864 --> 00:26:24,366 DIFFERENT LIGANDS, BUT THEN WE 718 00:26:24,366 --> 00:26:25,768 CHARACTERIZE THEM AT CP35 AS 719 00:26:25,768 --> 00:26:29,905 PROTEINS THAT ARE FUNCTIONING IN 720 00:26:29,905 --> 00:26:33,175 THE RECEPTOR RECYCLING AND WE 721 00:26:33,175 --> 00:26:35,277 FOUND THAT KCTA12 AND 2 PROTEINS 722 00:26:35,277 --> 00:26:40,249 ARE PROTEINS THAT REGULATE 723 00:26:40,249 --> 00:26:40,649 G-PROTEIN STASIS. 724 00:26:40,649 --> 00:26:42,785 SO SO NOW WE WANT TO GO TO THE 725 00:26:42,785 --> 00:26:44,653 NEXT STEP NOW THAT WE HAVE 726 00:26:44,653 --> 00:26:47,523 TESTED IN AND LIGANDS, HOW IS IT 727 00:26:47,523 --> 00:26:49,024 IF WE COMPARE DIFFERENT 728 00:26:49,024 --> 00:26:49,858 RECEPTORS, RIGHT? 729 00:26:49,858 --> 00:26:53,162 HOW SIMILAR ARE DIFFERENT DEC 730 00:26:53,162 --> 00:26:54,930 APPROXIMATE WE CAN MAP WITH OUR 731 00:26:54,930 --> 00:26:55,998 METHOD, SO THAT'S WHAT I WANT TO 732 00:26:55,998 --> 00:26:58,167 TALK ABOUT IN THE SECOND PART 733 00:26:58,167 --> 00:27:01,904 WHERE WE USE THE SAME APPROACH 734 00:27:01,904 --> 00:27:06,175 TO UNCOVER ANOTHER REGULATION OF 735 00:27:06,175 --> 00:27:07,576 GPCR SIGNALING TO ANOTHER 736 00:27:07,576 --> 00:27:07,843 RECEPTOR. 737 00:27:07,843 --> 00:27:10,112 SO TO START THIS UP RIGHT WE 738 00:27:10,112 --> 00:27:11,980 SAID OKAY, LET'S SELECT A NUMBER 739 00:27:11,980 --> 00:27:14,917 OF DIFFERENT RECEPTORS AND THEN 740 00:27:14,917 --> 00:27:18,120 THIS WAS PART OF A LARGER SCALE 741 00:27:18,120 --> 00:27:20,956 PROJECT THAT I STARTED AT UCSF 742 00:27:20,956 --> 00:27:22,591 IN COLLABORATION WITH US THAT 743 00:27:22,591 --> 00:27:24,693 WAS GEARED TOWARD FINDING NOVEL 744 00:27:24,693 --> 00:27:26,361 PAIN MEDICATION, SO THIS IS WHY 745 00:27:26,361 --> 00:27:29,498 THE RECEPTORS WE CHOSE HERE ARE 746 00:27:29,498 --> 00:27:30,632 RELATED SOMEHOW HAVE SOME 747 00:27:30,632 --> 00:27:33,268 FUNCTION IN PAIN REGULATION, 748 00:27:33,268 --> 00:27:35,904 HOWEVER, WHAT I'M TALKING ABOUT 749 00:27:35,904 --> 00:27:37,639 IS GENERAL BIOLOGY OF THE 750 00:27:37,639 --> 00:27:39,608 RECEPTOR, BUT THAT WAS A 751 00:27:39,608 --> 00:27:42,144 SELECTION CRITERIA, SO WE CHOSE 752 00:27:42,144 --> 00:27:42,978 DIFFERENT RECEPTOR FAMILIES 753 00:27:42,978 --> 00:27:44,646 AMONG THEM, THE OPIOID RECEPTOR, 754 00:27:44,646 --> 00:27:47,549 I TALKED ABOUT THE NEW OPh 755 00:27:47,549 --> 00:27:50,986 RECEPTOR BUT THERE ARE MULTIPLE 756 00:27:50,986 --> 00:28:01,430 RECEPTORS IN THIS FAMILY, 757 00:28:02,698 --> 00:28:04,233 RF-AMYDE, PROSTAGALA NDIN 758 00:28:04,233 --> 00:28:06,201 RECEPTORS, AND THEY HAVE KNOWN 759 00:28:06,201 --> 00:28:07,803 PROTEINS, AND THESE HAVE A 760 00:28:07,803 --> 00:28:09,204 DEFINITE LIGAND WE CHOOSE TO 761 00:28:09,204 --> 00:28:11,173 ACTIVATE AND SO WE HAVE 762 00:28:11,173 --> 00:28:11,974 DIFFERENT, DIFFERENT RECEPTOR 763 00:28:11,974 --> 00:28:13,542 FAMILIES AND THEY HAVE DIFFERENT 764 00:28:13,542 --> 00:28:15,277 FOR G-PROTEIN SO HOW THEN WE 765 00:28:15,277 --> 00:28:18,380 WERE WONDERING IF YOU USE OUR 766 00:28:18,380 --> 00:28:21,517 GPCR APEX APPROACH, HOW SIMILAR 767 00:28:21,517 --> 00:28:24,153 ON OTHER NETWORKS THAT THE 768 00:28:24,153 --> 00:28:26,021 RECEPTOR ENGAGES AND CAN WE USE 769 00:28:26,021 --> 00:28:27,289 THESE AGAIN AS WE FIND THINGS 770 00:28:27,289 --> 00:28:28,490 THAT ARE COMMON ACROSS 771 00:28:28,490 --> 00:28:30,759 RECEPTORS, BUT ALSO THINGS THAT 772 00:28:30,759 --> 00:28:32,427 LOOK DIFFERENT, RIGHT? 773 00:28:32,427 --> 00:28:34,296 AND AGAIN I SHOW YOU KIND OF BIG 774 00:28:34,296 --> 00:28:36,064 DATA BUT I DON'T GO THROUGH ALL 775 00:28:36,064 --> 00:28:40,235 OF THIS IN DETAIL SO WHEN WE 776 00:28:40,235 --> 00:28:41,537 LOOK HERE, THESE ARE THE RESULTS 777 00:28:41,537 --> 00:28:43,405 WE GET OUT FROM THE APEX 778 00:28:43,405 --> 00:28:46,074 APPROACH, THIS IS AGAIN A BIG 779 00:28:46,074 --> 00:28:47,042 HEAT MAP THAT DOESN'T SAY SO 780 00:28:47,042 --> 00:28:48,443 MUCH AND IMEAN I DON'T WANT TO 781 00:28:48,443 --> 00:28:49,745 GO THROUGH DETAILS OF ALL OF 782 00:28:49,745 --> 00:28:52,814 THIS, BUT I CAN SAY IS THAT WHEN 783 00:28:52,814 --> 00:28:54,516 WE LOOK ACROSS THE RECEPTORS, 784 00:28:54,516 --> 00:28:58,086 THERE ARE OBVIOUSLY SOME SIMILAR 785 00:28:58,086 --> 00:28:58,487 PATTERNS, RIGHT? 786 00:28:58,487 --> 00:29:00,355 WHICH IS NICE TO SEE AND THEY 787 00:29:00,355 --> 00:29:01,557 MAKE SENSE FOR EXAMPLE, WHEN WE 788 00:29:01,557 --> 00:29:02,858 LOOK AT THE 1S THAT ARE 789 00:29:02,858 --> 00:29:05,761 INDICATED, THESE ARE ALL THE 790 00:29:05,761 --> 00:29:09,998 G-RECEPTORS WHICH LEADS TO AN 791 00:29:09,998 --> 00:29:10,666 INCREASED IN INTRACELLULAR 792 00:29:10,666 --> 00:29:13,602 CALCIUM AND WHEN WOOK AT GENE 793 00:29:13,602 --> 00:29:14,803 ONTOLOGY ENRICHMENT OF THE 794 00:29:14,803 --> 00:29:16,905 PROTEINS IN DIFFERENT CLUSTERS 795 00:29:16,905 --> 00:29:20,008 WE SEE CALCIUM DEPENDENT PKC 796 00:29:20,008 --> 00:29:21,944 ACTIVITY IS ENRICHED FOR 797 00:29:21,944 --> 00:29:22,878 RECEPTORS THAT CAPPA HQ. 798 00:29:22,878 --> 00:29:25,514 WE SEE THIS SMIPGS AT THE 799 00:29:25,514 --> 00:29:30,686 EXPENSE, A LOT OF SIGNAL FOR 800 00:29:30,686 --> 00:29:31,753 ENDOCYTOSIS OR ENDOSOME FOR 801 00:29:31,753 --> 00:29:34,957 RECEPTORS AND WE KNOW UNDERGO 802 00:29:34,957 --> 00:29:35,691 TRAFFICKING LIKE INTERNALIZATION 803 00:29:35,691 --> 00:29:36,992 WHICH ALSO MAKES SENSE, BUT 1 804 00:29:36,992 --> 00:29:38,727 THING IS LIKE WHEN YOU LOOK AT 805 00:29:38,727 --> 00:29:39,928 THIS RIGHT, IT'S NOT ALWAYS 806 00:29:39,928 --> 00:29:41,263 THROUGH T-CELL WHAT YOU FALL OFF 807 00:29:41,263 --> 00:29:42,564 ON BUT 1 THING THAT WAS REALLY 808 00:29:42,564 --> 00:29:48,737 STRIKING TO US IS THAT THIS 809 00:29:48,737 --> 00:29:49,905 MPFS1, WHICH LOOKS ARE HAVE 810 00:29:49,905 --> 00:29:51,306 DIFFERENT THAN OTHER RECEPTORS 811 00:29:51,306 --> 00:29:52,808 HERE IN THIS HEAT MAP. 812 00:29:52,808 --> 00:29:58,013 IT'S A GI COUPLED RECEPTOR, 813 00:29:58,013 --> 00:29:59,414 INHIBITOR G-PROTEIN OPIOID 814 00:29:59,414 --> 00:30:03,585 RECEPTOR BUT THE PROFILE LOOKS 815 00:30:03,585 --> 00:30:04,653 COMPLETELY DIFFERENT IN THE 816 00:30:04,653 --> 00:30:05,153 OTHER RECEPTORS. 817 00:30:05,153 --> 00:30:07,055 AND WE SEE THERE'S SOME 818 00:30:07,055 --> 00:30:09,658 ENRICHMENT THAT IS SPECIFIC FOR 819 00:30:09,658 --> 00:30:12,427 MPP A 1, WHICH IS FOR THE LIGAND 820 00:30:12,427 --> 00:30:14,329 COMPLEX, SO NOW TO SIMPLIFY OUR 821 00:30:14,329 --> 00:30:16,832 RESULTS THERE, I WILL FOCUS JUST 822 00:30:16,832 --> 00:30:19,201 ON MPF 1 AND I WILL FOCUS ONLY 823 00:30:19,201 --> 00:30:21,003 ON THE PROTEINS THAT ARE SILG 95 824 00:30:21,003 --> 00:30:22,337 CANTILY CHANGING IN BIOTINNATION 825 00:30:22,337 --> 00:30:23,138 FOR THIS RECEPTOR. 826 00:30:23,138 --> 00:30:24,873 SO HERE THAT'S MUCH MORE SIMPLE 827 00:30:24,873 --> 00:30:27,075 TO LOOK AT, RIGHT IN WHETHER WE 828 00:30:27,075 --> 00:30:29,378 CAN SEE HERE WHEN WE FOCUS ON 829 00:30:29,378 --> 00:30:32,147 THIS IS THAT THERE'S A FEW 830 00:30:32,147 --> 00:30:33,582 PROTEINS IN STRONG CHANGES IN 831 00:30:33,582 --> 00:30:35,751 BIOTINNATION AND WHEN WE LOOK 832 00:30:35,751 --> 00:30:37,819 INTO THEM DEEPLY IS THAT THESE 833 00:30:37,819 --> 00:30:40,022 ARE -- THESE ARE MEMBERS OF THE 834 00:30:40,022 --> 00:30:41,523 SAME COMPLEX, SO THESE ARE 835 00:30:41,523 --> 00:30:46,561 MEMBERS OF A LIGAND, COLORING E3 836 00:30:46,561 --> 00:30:48,497 LIGANDS SO BTRC, AND THEY ARE 837 00:30:48,497 --> 00:30:50,265 SUBSTRATED THERE OF THIS 838 00:30:50,265 --> 00:30:51,600 COLORING HISTORY LIGAND COMPLEX, 839 00:30:51,600 --> 00:30:53,268 IT MEANS THEY BIND SUBSTRATES 840 00:30:53,268 --> 00:31:02,611 THAT ARE THEN BECOME 841 00:31:02,611 --> 00:31:03,245 UBIQUITINNATED, THE LIGASE ARE 842 00:31:03,245 --> 00:31:04,880 MODULAR, AND THEY ARE HIGHLY 843 00:31:04,880 --> 00:31:07,449 SIMILARITY IN TERMS OF THE 844 00:31:07,449 --> 00:31:09,318 SEQUENCE SPECIFICITY, THEY ARE 845 00:31:09,318 --> 00:31:10,452 BOTH SUBSTRATE WITH THIS. 846 00:31:10,452 --> 00:31:11,753 SO THIS WAS QUITE INTERESTING, 847 00:31:11,753 --> 00:31:13,622 WHEN YOU SEE SOMETHING LIKE 848 00:31:13,622 --> 00:31:15,090 THIS, YOU DON'T -- MULTIPLE 849 00:31:15,090 --> 00:31:16,091 MEMBERS OF THE SAME COMPLEX 850 00:31:16,091 --> 00:31:18,160 COMING UP IN A DATA SET THAT'S 851 00:31:18,160 --> 00:31:19,161 PROBABLY NOT HAPPENING BY CHANCE 852 00:31:19,161 --> 00:31:23,899 ISSUES - 853 00:31:23,899 --> 00:31:24,132 RIGHT? 854 00:31:24,132 --> 00:31:26,902 WE WANT TO VALIDATE IT AND 855 00:31:26,902 --> 00:31:28,303 RETURN IT WITH COMPLIMENTARY 856 00:31:28,303 --> 00:31:32,240 TECHNOLOGIES AND WE WORK CLOSELY 857 00:31:32,240 --> 00:31:34,176 WITH THE LAB AND IMAGING AND 858 00:31:34,176 --> 00:31:36,144 WHAT WE CAN SHOW WITH THEM HERE 859 00:31:36,144 --> 00:31:40,015 IS WHEN WE IMAGE THE -- WHEN WE 860 00:31:40,015 --> 00:31:41,783 IMAGE WE ACTIVATE THE RECEPTOR, 861 00:31:41,783 --> 00:31:46,688 WHEN WE HAVE THE NONACTIVATED 862 00:31:46,688 --> 00:31:49,391 RECEPTOR, THEN SBX W11 IS 863 00:31:49,391 --> 00:31:52,928 COMPUTE IN THE SIDE, WHEN WE 864 00:31:52,928 --> 00:31:55,597 ACTIVATE THE RECEPTOR WITH 865 00:31:55,597 --> 00:31:59,334 NPFFR1, IS THAT THE SUBSTRATE 866 00:31:59,334 --> 00:32:00,635 RECEPTOR REALLY CAN RELOCALLIZE 867 00:32:00,635 --> 00:32:03,038 TO THE PLASMA MEMBRANE AND CO 868 00:32:03,038 --> 00:32:05,741 LEGALAISE WITH THE RECEPTOR -- 869 00:32:05,741 --> 00:32:07,876 CO LOCALIZE WITH THE RECEPTOR. 870 00:32:07,876 --> 00:32:10,145 THIS ISN'T IN HEX CELLS BUT IS 871 00:32:10,145 --> 00:32:12,848 THIS SOMETHING CELL TYPE 872 00:32:12,848 --> 00:32:15,617 SPECIFIC, RIGHT? 873 00:32:15,617 --> 00:32:16,885 AND NPFFR1 IS ACTUALLY WHEN IT'S 874 00:32:16,885 --> 00:32:22,457 EXPRESSED IN CELLS THAT HAVE A 875 00:32:22,457 --> 00:32:23,892 PRIMARY ENTHELIUM, IT IS 876 00:32:23,892 --> 00:32:24,926 ENRICHED COMPARED TO THE PLAZ 877 00:32:24,926 --> 00:32:25,327 MIDS IN MEMBRANE. 878 00:32:25,327 --> 00:32:28,630 SO WHEN I SAY WE, THIS IS MARK'S 879 00:32:28,630 --> 00:32:35,003 LAB, WHAT WE COULD SHOW IS THAT 880 00:32:35,003 --> 00:32:37,172 THIS IS 1 THELIUM, AND IT'S REEL 881 00:32:37,172 --> 00:32:39,207 KEEPSAKESLY NICELY AND ENRICHES 882 00:32:39,207 --> 00:32:44,513 THE THELIUM CAN AND NOW WITHOUT 883 00:32:44,513 --> 00:32:47,149 ACTIVATION, FBXW11 IS IN THE 884 00:32:47,149 --> 00:32:48,884 BODY BUT YOU DON'T SEE IT IN THE 885 00:32:48,884 --> 00:32:50,852 THELLIAL CELLSIUM ITSELF AND 886 00:32:50,852 --> 00:32:52,954 WHEN IT'S ACTIVATED YOU SEE IT 887 00:32:52,954 --> 00:32:54,122 ENRICHED HERE AND CO LOCALIZED 888 00:32:54,122 --> 00:32:58,026 WITH THE RECEPTOR SO THIS IS 889 00:32:58,026 --> 00:32:59,294 SOMETHING THAT IS NOT CELL 890 00:32:59,294 --> 00:33:00,495 SPECIFIC WHICH IS GREAT, RIGHT? 891 00:33:00,495 --> 00:33:02,664 SO THE NEXT THOUGHT WE IS, WELL 892 00:33:02,664 --> 00:33:05,567 WE HAVE QUITE KNOWLEDGE ABOUT 893 00:33:05,567 --> 00:33:06,868 THESE E3 LIGASES, RIGHT? 894 00:33:06,868 --> 00:33:10,071 SO LIGASES THEY ARE RECRUITED TO 895 00:33:10,071 --> 00:33:12,040 A PROTEIN TO UBIQUITINNATE THE 896 00:33:12,040 --> 00:33:13,742 PROTEIN ITSELF OR SOMETHING IN 897 00:33:13,742 --> 00:33:14,910 THE SURROUNDING. 898 00:33:14,910 --> 00:33:21,049 SO WHAT WE KNOW FOR THIS 899 00:33:21,049 --> 00:33:24,886 SPECIFIC SUBSTRATE RECEPTOR, 900 00:33:24,886 --> 00:33:26,354 FBXW11 THEY RECOGNIZE THIS 901 00:33:26,354 --> 00:33:27,856 POSSIBLE DIAGRAM WITH THIS 902 00:33:27,856 --> 00:33:28,924 SEQUENCE AND WE WERE QUITE LUCKY 903 00:33:28,924 --> 00:33:30,659 BECAUSE FOR A LOT OF LIGASES 904 00:33:30,659 --> 00:33:31,526 THAT IS NOT THE FORM. 905 00:33:31,526 --> 00:33:36,031 SO WHEN WE LOOKED INTO THE 906 00:33:36,031 --> 00:33:37,532 RECEPTOR STRUCTURE THAT IS HERE 907 00:33:37,532 --> 00:33:38,600 OR THE RECEPTOR SEQUENCE, WE 908 00:33:38,600 --> 00:33:45,340 FOUND IN THE C-TERMINALS HERE, 909 00:33:45,340 --> 00:33:47,175 WE FOUND THIS SEQUENCE WHICH IS 910 00:33:47,175 --> 00:33:47,409 GREAT. 911 00:33:47,409 --> 00:33:49,911 SO FROM THIS FINDING THEN OUR 912 00:33:49,911 --> 00:33:52,013 HYPOTHESIS IS THAT THE RECEPTOR 913 00:33:52,013 --> 00:33:54,716 MIGHT BE PHOSPHORYLATED UPON 914 00:33:54,716 --> 00:33:56,218 ACTIVATION AND OUR GUESS WAS 915 00:33:56,218 --> 00:33:58,520 BECAUSE OF THE THE G-PROTEIN, 916 00:33:58,520 --> 00:34:02,524 THAT'S OUR FIRST GUESS, RIGHT, 917 00:34:02,524 --> 00:34:04,826 THESE ALSO PHOSPHORYLATE NPFFR1 918 00:34:04,826 --> 00:34:07,529 AND THEN THIS PHOSPHORYLATION OF 919 00:34:07,529 --> 00:34:09,831 THIS LEADS TO RECRUITMENT OF 920 00:34:09,831 --> 00:34:11,666 THESE 3 LIGAND COMPLEX, SO NOW 921 00:34:11,666 --> 00:34:14,769 IF ANYONE WANTS TO TEST THIS 922 00:34:14,769 --> 00:34:16,738 HYPOTHIS THERE IS 2 WAYS WE WENT 923 00:34:16,738 --> 00:34:18,373 ABOUT IT TO PERTURB THE SYSTEM. 924 00:34:18,373 --> 00:34:21,243 SO ON 1 HAND WE CAN MUTATE THE 925 00:34:21,243 --> 00:34:27,849 FOSTER NURSED FOCUSED ON 926 00:34:27,849 --> 00:34:28,216 FOSTER 927 00:34:28,216 --> 00:34:29,684 NURSED FOCUSED ON DEGRADATION 928 00:34:29,684 --> 00:34:30,819 RON, AND WE FOUND THAT WITH THE 929 00:34:30,819 --> 00:34:34,890 LIGASE AND ON THE OTHER HAND, 930 00:34:34,890 --> 00:34:37,792 THERE INHIBITORS FOR GRK, AND A 931 00:34:37,792 --> 00:34:40,829 GOOD INHIBITOR FOR GNK, 2 ASK 3, 932 00:34:40,829 --> 00:34:42,297 AND THIS 1 TARGETS SPECIFICALLY 933 00:34:42,297 --> 00:34:44,633 2 ANDLY AND TO BE HONEST WE JUST 934 00:34:44,633 --> 00:34:46,635 TARGET THIS BECAUSE THAT'S GOOD 935 00:34:46,635 --> 00:34:47,502 COMPOUND AVAILABLE, RIGHT? 936 00:34:47,502 --> 00:34:52,007 THIS IS THE WHAT WE DO, YOU HAVE 937 00:34:52,007 --> 00:34:53,542 AVAILABLE TOOL SO WE DID THIS. 938 00:34:53,542 --> 00:34:54,809 AND SOMETIMES BIOLOGY TURNS OUT 939 00:34:54,809 --> 00:34:56,678 TO BE TRUE AND KIND TO US, 940 00:34:56,678 --> 00:34:56,912 RIGHT? 941 00:34:56,912 --> 00:34:59,548 THIS EXPERIMENT, SO WHEN WE NOW 942 00:34:59,548 --> 00:35:00,715 COMPARED THE DIFFERENT 943 00:35:00,715 --> 00:35:02,684 CONDITION, WE PERFORMED THE 944 00:35:02,684 --> 00:35:04,152 APEX EXPERIMENT THEN FOR ACROSS 945 00:35:04,152 --> 00:35:05,921 THIS CONDITION FOR THE WILD-TYPE 946 00:35:05,921 --> 00:35:08,323 RECEPTOR FOR THE MUTANT TNT MA 947 00:35:08,323 --> 00:35:10,659 DOESN'T HAVE THE POSSIBLE DEGRON 948 00:35:10,659 --> 00:35:12,794 ANYMORE, AND ALSO THE 1 TREATED 949 00:35:12,794 --> 00:35:13,962 WITH COMPOUND 101. 950 00:35:13,962 --> 00:35:16,298 IF WE NOW SIMPLY LOOK AT THESE 951 00:35:16,298 --> 00:35:18,533 COMPONENTS OF THE LIGASE, WE SEE 952 00:35:18,533 --> 00:35:21,202 THAT THIS CHANGE IN 953 00:35:21,202 --> 00:35:22,804 BIOTINNATION, WHEN WE ACTIVATE 954 00:35:22,804 --> 00:35:24,239 THE WILD-TYPE, WITH OTHER 955 00:35:24,239 --> 00:35:25,840 CONDITIONS RIGHT, IT'S 956 00:35:25,840 --> 00:35:26,908 COMPLETELY BLOCKED, THE LIGASE 957 00:35:26,908 --> 00:35:28,443 IS NOT RECRUITED TO THE 958 00:35:28,443 --> 00:35:28,710 RECEPTOR. 959 00:35:28,710 --> 00:35:31,012 SO THIS WAS -- THIS WAS GREAT. 960 00:35:31,012 --> 00:35:33,081 SO WE THOUGHT, PROBABLY OUR 961 00:35:33,081 --> 00:35:35,684 HYPOTHESIS IS TRUE, RIGHT? 962 00:35:35,684 --> 00:35:39,521 AND THEN OF COURSE, WE WANTED TO 963 00:35:39,521 --> 00:35:41,022 PROVIDE IT AGAIN WITH A METHOD, 964 00:35:41,022 --> 00:35:44,025 ABOUT YOU WE ALSO THOUGHT, WELL, 965 00:35:44,025 --> 00:35:46,428 IF THIS PHOSPHORYLATION IS 966 00:35:46,428 --> 00:35:51,166 MEDIATE BY GRK, THEN THE 967 00:35:51,166 --> 00:35:53,101 CANNONICLE MODEL THEN MEDIATED 968 00:35:53,101 --> 00:35:55,537 PHOSPHORYLATION LEADS TO 969 00:35:55,537 --> 00:35:56,571 RECRUITMENT OF ARRESTINS, THE 970 00:35:56,571 --> 00:35:59,307 QUESTION S&P THIS INTERACTION 971 00:35:59,307 --> 00:36:00,575 DEPENDENT OR INDEPENDENT OF 972 00:36:00,575 --> 00:36:01,176 ARRESTIN? 973 00:36:01,176 --> 00:36:05,847 SO TO TEST THIS RIGHT THEN WE 974 00:36:05,847 --> 00:36:08,483 WORK WITH THE LAB, SPECIFICALLY 975 00:36:08,483 --> 00:36:09,317 A GRADUATE STUDENT IN THE LAB 976 00:36:09,317 --> 00:36:14,789 AND THEY HAVE THIS 9 977 00:36:14,789 --> 00:36:15,924 COMPLEMENTATION ASSAY CENTER 978 00:36:15,924 --> 00:36:17,659 WHERE WE HAVE 1 PART OF THE 979 00:36:17,659 --> 00:36:20,562 LARGE BIT, WE HAVE TARGET TO THE 980 00:36:20,562 --> 00:36:22,597 PLASMA MEMBRANE, AND THE SMALL 981 00:36:22,597 --> 00:36:24,566 BIT WE CAN TARGET TO ANY 982 00:36:24,566 --> 00:36:25,867 PROTEINS WE ARE INTERESTED IN. 983 00:36:25,867 --> 00:36:28,937 IN OUR CASE, WHAT WE EXPECT IS 984 00:36:28,937 --> 00:36:33,475 IF WE -- IF WE USE THE LIGASE, 985 00:36:33,475 --> 00:36:39,014 IF WE ACTIVATE OUR RECEPTOR THAT 986 00:36:39,014 --> 00:36:40,115 THE FAW11 RELOCALLIZE TO THE 987 00:36:40,115 --> 00:36:43,652 PLASMA MEMBRANE AND THEN WE GET 988 00:36:43,652 --> 00:36:44,519 THE LUMEN SIGNAL. 989 00:36:44,519 --> 00:36:46,821 SO WE NOW USE THIS IN A 990 00:36:46,821 --> 00:36:48,456 DIFFERENT CONDITION, SO WE 991 00:36:48,456 --> 00:36:50,892 AGAIN, WE TARGET THIS MODEL TO 992 00:36:50,892 --> 00:36:53,428 THE 11, WE INCLUDED WILD-TYPE 993 00:36:53,428 --> 00:36:54,863 RECEPTOR, INCLUDED THE MUTANT 994 00:36:54,863 --> 00:36:56,498 RECEPTOR THAT THE SHOW BEFORE 995 00:36:56,498 --> 00:36:58,099 AND A PROTEOMIC DATA THAT 996 00:36:58,099 --> 00:36:59,300 DOESN'T RECRUIT THE E3 LIGASE, 997 00:36:59,300 --> 00:37:01,536 BUT THEN WE ALSO WANTED TO HAVE 998 00:37:01,536 --> 00:37:03,705 THE NEW OPIOID RECEPTOR AS AN 999 00:37:03,705 --> 00:37:05,040 EXAMPLE OF CLASSIC RECEPTOR THEN 1000 00:37:05,040 --> 00:37:08,777 WHEN YOU ACTIVATE IT, IT 1001 00:37:08,777 --> 00:37:10,545 RECRUITS, IT'S PHOSPHORYLATED BY 1002 00:37:10,545 --> 00:37:11,646 GRK AND RECRUITS ARRESTIN. 1003 00:37:11,646 --> 00:37:13,381 SO JUST AS A COMPARISON, RIGHT? 1004 00:37:13,381 --> 00:37:16,051 AND WHAT WE CAN SHOW HERE OR THE 1005 00:37:16,051 --> 00:37:17,352 E3 LIGASE, IS AS SPECIFICALLY 1006 00:37:17,352 --> 00:37:18,386 FOR THE WILD-TYPE, IT DOESN'T 1007 00:37:18,386 --> 00:37:20,755 HAPPEN FOR THE MUTANT RECEPTOR 1008 00:37:20,755 --> 00:37:21,990 BUT IT ALSO DOESN'T HAVE -- 1009 00:37:21,990 --> 00:37:22,991 WHICH WE WOULD EXPECT. 1010 00:37:22,991 --> 00:37:27,328 NOW WE DO THE SAME EXPERIMENT, 1011 00:37:27,328 --> 00:37:29,698 WHERE WE TARGET THE SMALL, BUT 1012 00:37:29,698 --> 00:37:30,231 TO ARREST IT. 1013 00:37:30,231 --> 00:37:36,871 SO WHAT WE SEE HERE IS AN 1014 00:37:36,871 --> 00:37:39,040 OPPOSITE PICTURE, AND THE STRONG 1015 00:37:39,040 --> 00:37:41,209 LUMINESCE ENSEL SIGNAL, RIGHT? 1016 00:37:41,209 --> 00:37:42,610 BECAUSE THE ARRESTIN IS 1017 00:37:42,610 --> 00:37:44,112 RECRUITED THROUGH A REPRESENTOR 1018 00:37:44,112 --> 00:37:45,513 THROUGH ACTIVATION AND 1019 00:37:45,513 --> 00:37:46,347 POSFORALATION THROUGH GRK, BUT 1020 00:37:46,347 --> 00:37:48,983 NEITHER FOR THE WILD-TYPE TYPE, 1021 00:37:48,983 --> 00:37:50,785 FOR THE MUTANT RECEPTOR WE HAVE 1022 00:37:50,785 --> 00:37:51,352 ARRESTIN RECRUITMENT. 1023 00:37:51,352 --> 00:37:53,722 SO THIS IS VERY INTERESTING, 1024 00:37:53,722 --> 00:37:56,691 RIGHT WHAT WE CONCLUDE SO FAR 1025 00:37:56,691 --> 00:37:58,793 FROM THIS, IS THAT WE NOW HAVE, 1026 00:37:58,793 --> 00:38:02,330 WE DISCOVER A NOVEL INTERACTION 1027 00:38:02,330 --> 00:38:07,035 BETWEEN THE NPFFR1 RECEPTOR AND 1028 00:38:07,035 --> 00:38:09,471 THE 3 LIGASE, USING OUR 1029 00:38:09,471 --> 00:38:10,872 PROTEOMIC APPROACH AND BEING 1030 00:38:10,872 --> 00:38:11,573 LOAMACYING ACROSS DIFFERENT 1031 00:38:11,573 --> 00:38:14,075 RECEPTORS AND WHAT IS GREAT IS 1032 00:38:14,075 --> 00:38:16,711 THAT WE CAN SHOW THAT THESE 1033 00:38:16,711 --> 00:38:17,912 INTERACTIONS MEDIATE BY THE 1034 00:38:17,912 --> 00:38:20,482 FOSTER NURSED FOCUSED ON DEGRON, 1035 00:38:20,482 --> 00:38:22,484 OF THE TAIL OF THE RECEPTOR 1036 00:38:22,484 --> 00:38:23,685 WHICH IS PHOSPHORYLATED THROUGH 1037 00:38:23,685 --> 00:38:25,286 GRK AND INDEPENDENT OF THE 1038 00:38:25,286 --> 00:38:25,653 RESIDENCE. 1039 00:38:25,653 --> 00:38:27,122 OF COURSE NOW, THE BIG QUESTION 1040 00:38:27,122 --> 00:38:28,790 IS AND I AM SORRY I CANNOT GIVE 1041 00:38:28,790 --> 00:38:30,391 YOU THE ANSWER FOR IT BECAUSE 1042 00:38:30,391 --> 00:38:31,693 WE'RE STILL LOOKING AT THIS, 1043 00:38:31,693 --> 00:38:32,761 RIGHT, THE QUESTION IS WHAT THE 1044 00:38:32,761 --> 00:38:36,765 FUNCTION OF THIS RECRUITMENT, 1045 00:38:36,765 --> 00:38:36,965 RIGHT? 1046 00:38:36,965 --> 00:38:38,833 AND SO WE ARE LOOKING AT WHAT IS 1047 00:38:38,833 --> 00:38:41,302 THE EFFECT ON THE G-DEPENDENT 1048 00:38:41,302 --> 00:38:42,570 PROTEIN SIGNALING BUT ALSO 1049 00:38:42,570 --> 00:38:44,906 BECAUSE IT'S AN E3 LIGASE, AND 1050 00:38:44,906 --> 00:38:47,308 THE RECEPTOR, WHAT IS THE EFFECT 1051 00:38:47,308 --> 00:38:49,043 ON RECEPTOR'S ABILITY SO IT'S 1052 00:38:49,043 --> 00:38:51,279 SOMETHING THAT CONTINUED IN MY 1053 00:38:51,279 --> 00:38:52,747 LAB AND GOOD FOR FOLLOWING UP 1054 00:38:52,747 --> 00:38:53,114 ON. 1055 00:38:53,114 --> 00:38:53,681 YOU KNOW WHAT? 1056 00:38:53,681 --> 00:38:55,617 I STILL HAVE SOMETIME, RIGHT? 1057 00:38:55,617 --> 00:38:57,552 I WANT TO SHOW THE LAST PART 1058 00:38:57,552 --> 00:38:59,521 THAT IS IN PROGRESS NOW BECAUSE 1059 00:38:59,521 --> 00:39:01,122 WHAT WAS -- WHAT BECAME CLEAR TO 1060 00:39:01,122 --> 00:39:03,925 US BASED ON THIS EXAMPLE, IS 1061 00:39:03,925 --> 00:39:05,894 THAT MAYBE THERE'S MORE TO IT 1062 00:39:05,894 --> 00:39:07,862 THAN THIS CANNONICLE MODEL, 1063 00:39:07,862 --> 00:39:08,263 RIGHT? 1064 00:39:08,263 --> 00:39:10,064 THAT MAKES SENSE, RIGHT? 1065 00:39:10,064 --> 00:39:12,367 WHEN YOU THINK ABOUT GPC Rs 1066 00:39:12,367 --> 00:39:14,135 THEY HAVE A C-TERMINAL TAIL AND 1067 00:39:14,135 --> 00:39:15,637 SOMEHOW ALSO SOME OF THE LOOPS 1068 00:39:15,637 --> 00:39:17,739 CAN BE PRETTY LONG. 1069 00:39:17,739 --> 00:39:18,773 THEY'RE VERY UNSTRUCTURED, 1070 00:39:18,773 --> 00:39:19,207 RIGHT? 1071 00:39:19,207 --> 00:39:25,013 YOU HAVE PHOSPHORYLATION OF 1072 00:39:25,013 --> 00:39:27,182 THESE AUTOSECRETION, THEY CAN 1073 00:39:27,182 --> 00:39:28,116 HAVE CONDITIONING FOR THE 1074 00:39:28,116 --> 00:39:30,552 SITUATION WHERE THEY CAN ENGAGE 1075 00:39:30,552 --> 00:39:31,820 PROTEINS AND SOMEHOW, THIS SEEMS 1076 00:39:31,820 --> 00:39:33,822 VERY SIMPLE TO THINK THERE ARE 1077 00:39:33,822 --> 00:39:35,657 MULTIPLE GRKs THAT CAN 1078 00:39:35,657 --> 00:39:36,491 PHOSPHORYLATE THE DIFFERENT 1079 00:39:36,491 --> 00:39:38,493 RECEPTOR TAILS AND THESE TAILS 1080 00:39:38,493 --> 00:39:40,428 CAN BE QUITE DEFINITE IN TERMS 1081 00:39:40,428 --> 00:39:41,763 OF LENGTH AND SEQUENCE AND THEY 1082 00:39:41,763 --> 00:39:43,264 MIGHT RECRUIT OTHER PROEN TOOS 1083 00:39:43,264 --> 00:39:45,133 THAT ARE ONLY ARRESTINS, RIGHT 1084 00:39:45,133 --> 00:39:47,235 AND THIS IS NOT ONLY BASED ON 1085 00:39:47,235 --> 00:39:49,437 OUR FINDINGS, SO THIS IS THE 1086 00:39:49,437 --> 00:39:54,342 CLASSIC MODEL HERE, RIGHT, THE 1087 00:39:54,342 --> 00:39:56,077 GRK PHOSPHORYLATOR ARRESTIN AND 1088 00:39:56,077 --> 00:39:57,512 THEN THE PHOSPHORYLATION AND 1089 00:39:57,512 --> 00:39:58,379 MEDIATION. 1090 00:39:58,379 --> 00:40:00,915 BUT THERE'S MUCH MORE EVIDENCE 1091 00:40:00,915 --> 00:40:03,952 EMERGE NOTHING LITERATURE THAT 1092 00:40:03,952 --> 00:40:05,053 THERE'S GRK REGULATION AND 1093 00:40:05,053 --> 00:40:08,122 RECRUITMENT SO WE FOUND THIS 1094 00:40:08,122 --> 00:40:11,593 INTERACTION OF THE NPFFR1, AND 1095 00:40:11,593 --> 00:40:16,197 INDEPENDENT OF ARRESTIN AND IT'S 1096 00:40:16,197 --> 00:40:18,132 BEAUTIFUL WORK BY BEN WHO HAS 1097 00:40:18,132 --> 00:40:21,336 SHOWN THAT USING THE RECEPTOR IS 1098 00:40:21,336 --> 00:40:23,071 IMPORTANT FOR DEVELOPMENT, WHEN 1099 00:40:23,071 --> 00:40:24,372 ACTIVATED IT PHOSPHORYLATES BY 1100 00:40:24,372 --> 00:40:27,041 GRK WHICH LEADS NOT TO THE 1101 00:40:27,041 --> 00:40:29,477 RECRUITMENT OF ARRESTIN, BUT THE 1102 00:40:29,477 --> 00:40:30,879 SUBUNIT OF PKAA, AND I HAVE 1103 00:40:30,879 --> 00:40:33,748 SHOWN NICELY THIS IS A KEY EVENT 1104 00:40:33,748 --> 00:40:42,090 TO REGULATE TRANSCRIPTION DOWN 1105 00:40:42,090 --> 00:40:43,625 STREAM OF FOX. 1106 00:40:43,625 --> 00:40:44,659 AND IT'S BASED CONDITIONALLY ON 1107 00:40:44,659 --> 00:40:46,628 THE SEQUENCE OF THE C-TERMINUS 1108 00:40:46,628 --> 00:40:47,862 TAIL AND THEN THERE ARE A NUMBER 1109 00:40:47,862 --> 00:40:49,497 OF STUDIES SHOWN FOR A NUMBER OF 1110 00:40:49,497 --> 00:40:51,165 RECEPTORS THAT ARE KNOWN TO 1111 00:40:51,165 --> 00:40:52,200 INTERNALIZE AND ARRESTIN IN AN 1112 00:40:52,200 --> 00:40:53,268 INDEPENDENT WAY. 1113 00:40:53,268 --> 00:40:54,569 SO WE THOUGHT WHILE THIS 1114 00:40:54,569 --> 00:40:57,171 EMERGING DATA IN THE LITERATURE, 1115 00:40:57,171 --> 00:41:00,675 EXAMPLES, CAN BE MAY BE USE OUR 1116 00:41:00,675 --> 00:41:02,644 PROTEOMIC APPROACH TO IN A 1117 00:41:02,644 --> 00:41:05,613 LARGER SCALE TO EXPLORE 1 GRK 1118 00:41:05,613 --> 00:41:06,447 FUNCTION BEYOND ARRESTIN 1119 00:41:06,447 --> 00:41:08,616 RECRUITMENT IN THE SYSTEMATIC 1120 00:41:08,616 --> 00:41:10,518 MANNER, AND IT'S ACTUALLY QUITE 1121 00:41:10,518 --> 00:41:14,188 AN UNDERTAKING SO I HAD THIS 1122 00:41:14,188 --> 00:41:15,256 IDEA, MAYBE SEVERAL MONTHS AGO 1123 00:41:15,256 --> 00:41:16,791 AND I THOUGHT IF WE EMBARK ON 1124 00:41:16,791 --> 00:41:18,092 THIS, WE HAVE TO DESIGN THIS 1125 00:41:18,092 --> 00:41:20,061 REALLY WELL AND WHAT YOU ALSO 1126 00:41:20,061 --> 00:41:23,598 NEED IS LIKE A STUDENT WHO IS UP 1127 00:41:23,598 --> 00:41:24,565 FOR A BIGGER CHALLENGE. 1128 00:41:24,565 --> 00:41:26,100 SO THIS IS SOMETHING THAT WE ARE 1129 00:41:26,100 --> 00:41:27,835 GEARING UP TOWARDS NOW AND I 1130 00:41:27,835 --> 00:41:29,704 JUST WANT TO SHOW YOU JUST TELL 1131 00:41:29,704 --> 00:41:31,873 YOU HOW WE'RE THINKING OF DOING 1132 00:41:31,873 --> 00:41:33,541 IT AND BITS AND PIECES OF A 1133 00:41:33,541 --> 00:41:35,410 LITTLE BIT OF DATA, NOT TOO MUCH 1134 00:41:35,410 --> 00:41:36,811 BUT JUST TO GIVE YOU A SENSE OF 1135 00:41:36,811 --> 00:41:38,780 WHAT I HOPE WE CAN FIND FROM 1136 00:41:38,780 --> 00:41:39,914 THIS. 1137 00:41:39,914 --> 00:41:42,450 SO OUR IDEAS, MAYBE A BIT CRAZY 1138 00:41:42,450 --> 00:41:44,085 BUT I THINK THE STRENGTH COMES 1139 00:41:44,085 --> 00:41:47,689 IN NUMBERS, SO WE -- WE CHOOSE A 1140 00:41:47,689 --> 00:41:48,957 NUMBER OF RECEPTORS, RIGHT IN 1141 00:41:48,957 --> 00:41:50,692 AND OUR INITIAL TARGET IS MAYBE 1142 00:41:50,692 --> 00:41:52,260 50, AND THE QUESTION IS HOW DO 1143 00:41:52,260 --> 00:41:53,628 WE SELECT THEM, RIGHT? 1144 00:41:53,628 --> 00:41:56,464 SO WE THOUGHT, WE CAN MAKE THE 1145 00:41:56,464 --> 00:41:59,500 SELECTION BASED ON A DIFFERENT 1146 00:41:59,500 --> 00:42:01,602 LENGTH AND COMCISION OF 1147 00:42:01,602 --> 00:42:02,403 C-TERMINAL TAILS, RIGHT? 1148 00:42:02,403 --> 00:42:06,574 THEN OF COURSE, WE SELECT ONLY 1149 00:42:06,574 --> 00:42:08,209 RECEPTORS THAT ARE NOT WORKING 1150 00:42:08,209 --> 00:42:11,746 SO YOU HAVE NEGATIVE SELECTION, 1151 00:42:11,746 --> 00:42:13,314 AND THEN, YOU KNOW WHAT WE 1152 00:42:13,314 --> 00:42:15,783 ACTUALLY FOUND QUITE INTERESTING 1153 00:42:15,783 --> 00:42:16,884 IS THAT ALPHA FOLD IS NOT GOOD 1154 00:42:16,884 --> 00:42:18,086 FOR A NUMBER OF STRUCTURES AND 1155 00:42:18,086 --> 00:42:20,455 WE DON'T KNOW HOW GOOD IT IS, 1156 00:42:20,455 --> 00:42:27,395 BUT FOR A SPECIFIC EXAMPLE OF 1157 00:42:27,395 --> 00:42:28,796 SMOOTHNESS, IT CAN PREDICT 1158 00:42:28,796 --> 00:42:29,731 CONDITIONALLY FOR THE STATE. 1159 00:42:29,731 --> 00:42:32,367 AND WHAT WAS FOUND IN THE 1160 00:42:32,367 --> 00:42:34,902 EXAMPLE OF SMOOTHING WHERE IT 1161 00:42:34,902 --> 00:42:37,939 ENGAGES THE PK,A,A, THAN ALPHA 1162 00:42:37,939 --> 00:42:39,674 FOLD PREDICT THIS SHORT ALPHA 1163 00:42:39,674 --> 00:42:42,110 HELIX THERE WHICH ACTUALLY BINDS 1164 00:42:42,110 --> 00:42:44,178 CATALYTIC SUBUNIT OF PKAA AND WE 1165 00:42:44,178 --> 00:42:47,682 THOUGHT WE COULD ALSO USE ALPHA 1166 00:42:47,682 --> 00:42:50,485 FOLD THEN, TO FIND DIFFERENT 1167 00:42:50,485 --> 00:42:51,285 REGIONS ACROSS RECEPTORS, 1168 00:42:51,285 --> 00:42:54,655 DIFFERENT REGION OF THE 1169 00:42:54,655 --> 00:42:56,024 C-TERMINUS TAIL, AND AT THE END 1170 00:42:56,024 --> 00:42:57,325 WE ARE TESTING THIS HYPOTHESIS. 1171 00:42:57,325 --> 00:42:59,160 SO THIS IS THE WAY THAT WE 1172 00:42:59,160 --> 00:42:59,794 APPROACH THIS. 1173 00:42:59,794 --> 00:43:02,030 NOW I CANNOT TELL YOU IF THAT'S 1174 00:43:02,030 --> 00:43:03,431 A VARIED APPROACH OR NOT, BUT WE 1175 00:43:03,431 --> 00:43:06,401 CHOOSE THEM WHERE WE SEE 1176 00:43:06,401 --> 00:43:07,535 INTERESTING ALPHA HELICAL 1177 00:43:07,535 --> 00:43:09,370 PREDICTIONS AND SOME WHERE WE 1178 00:43:09,370 --> 00:43:10,338 DON'T SEE PREDICTION AND SEE 1179 00:43:10,338 --> 00:43:11,406 WHAT COMES OUT OF OUR STUDY. 1180 00:43:11,406 --> 00:43:14,842 SO THIS IS HOW WE,A PROACH NOW 1181 00:43:14,842 --> 00:43:16,344 THE SELECTION OF THE TCR. 1182 00:43:16,344 --> 00:43:17,678 THEN WHAT WE THOUGHT IS INSTEAD 1183 00:43:17,678 --> 00:43:20,081 OF JUST DOING THE APEX 1184 00:43:20,081 --> 00:43:21,682 EXPERIMENT IN WILD-TYPE CELLS, 1185 00:43:21,682 --> 00:43:23,651 WHAT WOULD BE ACTUALLY VERY 1186 00:43:23,651 --> 00:43:25,720 INFORMATIVE IF WE DIRECT USE 3 1187 00:43:25,720 --> 00:43:27,488 DIFFERENT FLAVORS OF CELL LINES, 1188 00:43:27,488 --> 00:43:29,490 WE USE WILD-TYPE CELLS, WE USELE 1189 00:43:29,490 --> 00:43:31,159 CELLS THAT HAVE ARRESTINS 1190 00:43:31,159 --> 00:43:32,393 COMPLETELY KNOCKED OUT SO THAT 1191 00:43:32,393 --> 00:43:34,262 WE CAN SEE OTHER INTERACTIONS 1192 00:43:34,262 --> 00:43:37,432 THAT ARE DEPENDENT ON ARRESTINS 1193 00:43:37,432 --> 00:43:40,068 AND THEN -- AND THE 1 THAT HAS 1194 00:43:40,068 --> 00:43:41,869 GRK KNOCK OUT SO WE CAN SEE 1195 00:43:41,869 --> 00:43:44,272 THESE ON GRKs AND I THINK 1196 00:43:44,272 --> 00:43:46,307 WHILE THIS TROUBLE THE AMOUNT OF 1197 00:43:46,307 --> 00:43:48,176 WORK UPFRONT WE CAN GET A LOT OF 1198 00:43:48,176 --> 00:43:49,811 INFORMATION FROM IT UPFRONT AND 1199 00:43:49,811 --> 00:43:51,746 I TOLD YOU WE HAVE THE 1200 00:43:51,746 --> 00:43:53,281 ENRICHMENT SO WE THOUGHT WE ARE 1201 00:43:53,281 --> 00:43:55,616 GEARED SO IT MIGHT BE FEASIBLE, 1202 00:43:55,616 --> 00:43:58,453 RIGHT BUT THEN WHEN YOU WANT TO 1203 00:43:58,453 --> 00:44:00,555 PUT 50 GPCR IN THE 3 DIFFERENT 1204 00:44:00,555 --> 00:44:01,522 CELLS, THAT'S QUITE AN 1205 00:44:01,522 --> 00:44:02,790 UNDERTAKING SO WE THOUGHT TO 1206 00:44:02,790 --> 00:44:05,393 ALSO FIND AN ELEGANT APPROACH 1207 00:44:05,393 --> 00:44:10,465 HOW WE CAN DO THIS IN THE 1208 00:44:10,465 --> 00:44:12,066 MOLECULAR BIOLOGY WAY, SO WE 1209 00:44:12,066 --> 00:44:13,134 INTEGRATE THESE LANDING PAD INTO 1210 00:44:13,134 --> 00:44:15,002 THE 3 CELL LINES AND WHAT I 1211 00:44:15,002 --> 00:44:17,405 MEAN, THIS LANDING PAD WHEN WE 1212 00:44:17,405 --> 00:44:18,739 CAN EASY SWAP IN AND OUT 1213 00:44:18,739 --> 00:44:20,241 RECEPTORS OF THIS AND WE HAVE 1214 00:44:20,241 --> 00:44:23,811 BEEN DOING THAT AND MAKING THESE 1215 00:44:23,811 --> 00:44:24,278 CELL CHARACTERIZATION. 1216 00:44:24,278 --> 00:44:27,582 AND THIS IS WHAT WE HAVE 1217 00:44:27,582 --> 00:44:28,015 CURRENTLY SET UP. 1218 00:44:28,015 --> 00:44:31,085 SO THE NEXT STEPS ARE THEN OF 1219 00:44:31,085 --> 00:44:36,824 COURSE TO DO THE GPCR SELTZMENT 1220 00:44:36,824 --> 00:44:38,993 AND AGAIN, WHAT I WAS HOPING IS 1221 00:44:38,993 --> 00:44:40,194 THAT THE DATA WOULD LIKE LIKE 1222 00:44:40,194 --> 00:44:41,028 THIS A LITTLE BIT. 1223 00:44:41,028 --> 00:44:43,498 WE HAVE THEM WHEN WE LOOK AT 1224 00:44:43,498 --> 00:44:46,267 ACROSS A DIFFERENT CELL LINE AND 1225 00:44:46,267 --> 00:44:47,635 RECEPTORS, WE CAN FIND 1226 00:44:47,635 --> 00:44:50,171 INTERACTION THAT IS POST GRK AND 1227 00:44:50,171 --> 00:44:52,039 THE ARRESTIN INDEPENDENT OR GRK 1228 00:44:52,039 --> 00:44:54,575 DEPENDENT SO BY THIS, RIGHT IN 1229 00:44:54,575 --> 00:44:55,810 THIS IS HELPFUL. 1230 00:44:55,810 --> 00:44:57,111 BEFORE YOU -- BEFORE YOU EMBARK 1231 00:44:57,111 --> 00:44:59,947 ON THIS HUGE STUDY, YOU WANT TO 1232 00:44:59,947 --> 00:45:02,917 ACTUALLY SHOW THAT THIS IS 1233 00:45:02,917 --> 00:45:08,022 ACTUALLY TRUE FOR SOME -- FOR 1234 00:45:08,022 --> 00:45:11,325 PRIOR KNOWLEDGE, SO JUST AS A 1235 00:45:11,325 --> 00:45:15,429 SANITY CHECK RIGHT WE DID THIS 1236 00:45:15,429 --> 00:45:17,698 FOR NFPPR1, AND WE HAVE THIS 1237 00:45:17,698 --> 00:45:19,667 INTERACTION OF THE LIGATES 1238 00:45:19,667 --> 00:45:21,502 INDEPENDENT OF ARRESTIN AND WE 1239 00:45:21,502 --> 00:45:22,503 DID THE APEX EXPERIMENT ACROSS 1240 00:45:22,503 --> 00:45:24,105 THE 3 CELL LINES AND WHAT WAS 1241 00:45:24,105 --> 00:45:25,406 REALLY STRIKING TO SEE, I 1242 00:45:25,406 --> 00:45:27,141 THOUGHT THIS WAS SO GOOD TO SEE, 1243 00:45:27,141 --> 00:45:28,809 YOU DON'T EVEN NEED STATISTICS 1244 00:45:28,809 --> 00:45:30,778 FOR THIS, IS REALLY THE E3 1245 00:45:30,778 --> 00:45:32,313 LIGASE COMOPPOSITE BEHAVIORIAL 1246 00:45:32,313 --> 00:45:32,880 PHENOTYPENTS THAT COMPLETELY 1247 00:45:32,880 --> 00:45:34,515 DISAPPEAR IN THE GRK KNOCK OUT 1248 00:45:34,515 --> 00:45:36,150 CELL LINE WHEN WE HAVE THE 1249 00:45:36,150 --> 00:45:38,252 ARRESTIN KNOCK OUT, THEY LOOK 1250 00:45:38,252 --> 00:45:40,121 BASICALLY IDENTICAL, AND THEN WE 1251 00:45:40,121 --> 00:45:41,322 SEE ALSO DOWN STREAM 1252 00:45:41,322 --> 00:45:43,491 INTERACTION, SO FOR EXAMPLE, 1253 00:45:43,491 --> 00:45:45,826 HERE, THIS IS -- THESE ARE ALL 1254 00:45:45,826 --> 00:45:49,397 MEMBERS OF THE ESCORT LIKE 1255 00:45:49,397 --> 00:45:52,033 COMPLEX, THAT ARE LIKE UBIQUITIN 1256 00:45:52,033 --> 00:45:54,602 READ OUTS SO THE HYPOTHESIS FROM 1257 00:45:54,602 --> 00:45:55,236 THIS EXPERIMENT, AND YOU SEE 1258 00:45:55,236 --> 00:45:57,805 WHEN YOU LOOK AT THE TIME SCALE, 1259 00:45:57,805 --> 00:45:58,839 SEQUENT QUALLY THEY COME UP 1260 00:45:58,839 --> 00:46:02,043 LATER ABOUT YOU THEY'RE ALSO PDK 1261 00:46:02,043 --> 00:46:03,778 DEPENDENT SO OUR HYPOTHESIS IS 1262 00:46:03,778 --> 00:46:09,350 THAT THE LIGASE HERE IS 1263 00:46:09,350 --> 00:46:13,254 UBIQUITINNATING THE RECEPTOR AND 1264 00:46:13,254 --> 00:46:15,122 WE ARE VALIDATING AND I THINK WE 1265 00:46:15,122 --> 00:46:16,490 ALSO DID A SIMILAR EXPERIMENT, 1266 00:46:16,490 --> 00:46:19,393 AND WE ALSO GOT THERE, GRK 1267 00:46:19,393 --> 00:46:19,961 ARRESTIN INDEPENDENT 1268 00:46:19,961 --> 00:46:20,795 INTERACTION, LOOKS AT THE 1269 00:46:20,795 --> 00:46:21,862 DIFFERENT 1 HERE AND I THINK IF 1270 00:46:21,862 --> 00:46:23,898 WE HAVE THIS KIND OF DATA ACROSS 1271 00:46:23,898 --> 00:46:25,766 THE RECEPTORS WE CAN ACTUALLY 1272 00:46:25,766 --> 00:46:28,069 COME UP WITH REALLY NICE 1273 00:46:28,069 --> 00:46:29,437 HYPOTHESIS TO FOLLOW UP BUT WE 1274 00:46:29,437 --> 00:46:31,305 CAN MAYBE THEN ALSO, MY HOPE IS 1275 00:46:31,305 --> 00:46:33,307 RIGHT, WE CAN GO BACK TO LOOKING 1276 00:46:33,307 --> 00:46:35,343 INTO THE SEQUENCES OF THE 1277 00:46:35,343 --> 00:46:36,777 C-TERMINALS REPAIR OF THE 1278 00:46:36,777 --> 00:46:39,213 RECEPTOR AND FIND SOME SORT OF 1279 00:46:39,213 --> 00:46:39,914 PAIR UP, MAYBE. 1280 00:46:39,914 --> 00:46:41,749 SO THIS WAS ONGOING, BIG EFFORT 1281 00:46:41,749 --> 00:46:44,218 BUT I'M SUPER EXCITED FOR THE 1282 00:46:44,218 --> 00:46:44,885 RECEPTOR I'M SO EXCITED TO SEE 1283 00:46:44,885 --> 00:46:49,790 THE DAILY BASIS THEA -- DATA AD 1284 00:46:49,790 --> 00:46:51,325 SEE IF WE CAN SEE HOW THIS 1285 00:46:51,325 --> 00:46:51,993 PANNED OUT. 1286 00:46:51,993 --> 00:46:53,094 THE LAST BIT OF DAT A WANT TO 1287 00:46:53,094 --> 00:46:54,862 SHOW YOU AND IT'S REALLY ONLY 1 1288 00:46:54,862 --> 00:46:56,931 SLIDE, I SEE THAT LUCKILY WE ARE 1289 00:46:56,931 --> 00:46:59,233 GOOD ON TIME IS THAT RIGHT NOW 1290 00:46:59,233 --> 00:47:04,572 ALL OF THE SLIDES I TELL YOU IS 1291 00:47:04,572 --> 00:47:05,740 IN HEX-STYLE, BECAUSE WHEN WE 1292 00:47:05,740 --> 00:47:08,376 THINK ABOUT THIS SCREEN, WE CAN 1293 00:47:08,376 --> 00:47:10,111 PERFORM THIS EXPERIMENT IN 12 1294 00:47:10,111 --> 00:47:12,313 PERFORMANCES OR WE CAN LIKE THE 1295 00:47:12,313 --> 00:47:14,682 CELL CULTURE AND THEN THE THERE 1296 00:47:14,682 --> 00:47:16,450 WILD-TYPE, SO CAN YOU ACTUALLY 1297 00:47:16,450 --> 00:47:18,286 SCALE IT IN TERMS OF CONDITIONS 1298 00:47:18,286 --> 00:47:20,655 QUITE WELL. 1299 00:47:20,655 --> 00:47:23,190 AND THAT'S IN HEK THAT'S 1300 00:47:23,190 --> 00:47:24,725 FEASIBLE BUT OF COURSE, WHEN YOU 1301 00:47:24,725 --> 00:47:26,794 THINK ABOUT THESE RECEPTORS THEY 1302 00:47:26,794 --> 00:47:31,299 ARE EXPRESSED IN MANY DIFFERENT 1303 00:47:31,299 --> 00:47:32,933 CELL TYPES AND THIS 1304 00:47:32,933 --> 00:47:34,001 PASTEURIZATION CAN ALSO COME 1305 00:47:34,001 --> 00:47:35,169 FROM THE SIGNALING IN THE 1306 00:47:35,169 --> 00:47:36,904 EXPRESSED CELLS AND THAT'S WE 1307 00:47:36,904 --> 00:47:41,108 ARE WORKING TOWARDS RIGHT TO USE 1308 00:47:41,108 --> 00:47:42,043 MORE SPECIALIZED CELLS AND WHAT 1309 00:47:42,043 --> 00:47:44,679 I WANT TO JUST SHOW, THIS KIND 1310 00:47:44,679 --> 00:47:46,747 OF IMAGE AND I HAVE THE PICTURE 1311 00:47:46,747 --> 00:47:48,382 OF MIKE HERE IN THE AUDIENCE, SO 1312 00:47:48,382 --> 00:47:49,917 WE ARE RIGHT NOW WHAT WE ARE 1313 00:47:49,917 --> 00:47:52,253 SETTING UP, WE HAVE SET IT UP IN 1314 00:47:52,253 --> 00:47:54,822 THE LAB, OUR IPSC DIRECT NEURONS 1315 00:47:54,822 --> 00:47:57,358 WHICH WE ARE EXCITED TO USE FOR 1316 00:47:57,358 --> 00:48:00,461 THIS STUDY, FOR THE NEURONAL 1317 00:48:00,461 --> 00:48:01,529 RECEPTORS AND WE USE A SYSTEM 1318 00:48:01,529 --> 00:48:03,497 THAT WAS REALLY NICELY 1319 00:48:03,497 --> 00:48:06,901 IMPLEMENTED BY MICHAEL WHERE WE 1320 00:48:06,901 --> 00:48:09,970 HAVE THIS INDUCIBLE 1321 00:48:09,970 --> 00:48:11,806 TRANSCRIPTION FACTOR NG N2, AND 1322 00:48:11,806 --> 00:48:14,375 CAN LEAD TO YOU KNOW CORTICALE 1323 00:48:14,375 --> 00:48:17,044 NEURONS WITHIN 21 DAYS AND PLUS, 1324 00:48:17,044 --> 00:48:17,945 AND THAT'S JUST AN IMAGE OF 1325 00:48:17,945 --> 00:48:20,014 THESE CELLS AND I HOPE IN A FEW 1326 00:48:20,014 --> 00:48:23,684 MONTHS I CAN SHOW SOME OF THE 1327 00:48:23,684 --> 00:48:24,952 PROTEOMIC DATA WITHIN ANY CELL, 1328 00:48:24,952 --> 00:48:26,354 ANY CELL SYSTEM AND IT'S A LOT 1329 00:48:26,354 --> 00:48:29,023 OF WORK BY ANGELINNA IN THE LAB 1330 00:48:29,023 --> 00:48:30,825 AND COULD ONLY BE POSSIBLE 1331 00:48:30,825 --> 00:48:32,259 BECAUSE THIS IS STEEP LEARNING 1332 00:48:32,259 --> 00:48:33,494 CURVE BECAUSE IN THE LAB WE HAVE 1333 00:48:33,494 --> 00:48:34,829 NEVER DONE IT BUT MICHAEL HAS 1334 00:48:34,829 --> 00:48:36,564 BEEN SHARING A LOT OF -- A LOT 1335 00:48:36,564 --> 00:48:38,132 OF HIS KNOWLEDGE TO GET THIS UP 1336 00:48:38,132 --> 00:48:39,667 AND RUNNING IN THE LAB, SO THANK 1337 00:48:39,667 --> 00:48:41,435 YOU MICHAEL FOR THIS, AND I AM 1338 00:48:41,435 --> 00:48:43,838 REALLY EXCITED WE CAN USE THIS 1339 00:48:43,838 --> 00:48:45,039 CELL, PROBABLY NOT AS BIG, BUT 1340 00:48:45,039 --> 00:48:46,807 WE HAVE TO SCALE UP A BIT BUT 1341 00:48:46,807 --> 00:48:49,443 I'M VERY POSITIVE THAT WE CAN -- 1342 00:48:49,443 --> 00:48:50,845 WE CAN MOVE TOWARDS THIS CELL 1343 00:48:50,845 --> 00:48:51,812 SYSTEM, RIGHT IN AND IF YOU 1344 00:48:51,812 --> 00:48:56,050 THINK ABOUT IT, ONCE WE HAVE THE 1345 00:48:56,050 --> 00:48:56,984 DERIVED NEURONS BUT IF YOU'RE 1346 00:48:56,984 --> 00:49:00,688 ABLE TO DO THAT IN IPSC DERIVED 1347 00:49:00,688 --> 00:49:03,357 CELLS THEN YOU CAN INTEGRATE OUR 1348 00:49:03,357 --> 00:49:06,093 RECEPTOR OF INTEREST IN IPSCs 1349 00:49:06,093 --> 00:49:07,595 AND DIFFERENTIATE THEM INTO 1350 00:49:07,595 --> 00:49:09,130 DIFFERENT CELLS, AND LOOK ACROSS 1351 00:49:09,130 --> 00:49:10,931 DIFFERENT CELL TYPES SIGNALING. 1352 00:49:10,931 --> 00:49:12,466 SO THIS IS THE -- THIS IS THE 1353 00:49:12,466 --> 00:49:14,769 IDEA FOR THE FUTURE. 1354 00:49:14,769 --> 00:49:16,837 SO AGAIN, THIS IS THE VERSION 1355 00:49:16,837 --> 00:49:19,774 THERE I SHOWED YOU BEFORE THAT 1356 00:49:19,774 --> 00:49:20,975 WE ARE TRYING TO FOLLOW IN THE 1357 00:49:20,975 --> 00:49:23,778 LAB. 1358 00:49:23,778 --> 00:49:27,681 WE WANT TO STUDY WHAT DRIVES 1359 00:49:27,681 --> 00:49:29,884 SPECIFICITY OF GPCR SIGNALING 1360 00:49:29,884 --> 00:49:31,152 ACROSS DIFFERENT RECEPTORS, 1361 00:49:31,152 --> 00:49:32,520 ACROSS DIFFERENT LIGANDS BY 1362 00:49:32,520 --> 00:49:34,655 SIMPLY GENERATING DATA IN AN 1363 00:49:34,655 --> 00:49:36,991 EDUCATED WAY, RIGHT? 1364 00:49:36,991 --> 00:49:38,292 AND CORRELATING THIS WITH WITHIN 1365 00:49:38,292 --> 00:49:39,693 THE FUTURE WITH INVITRO 1366 00:49:39,693 --> 00:49:41,128 PHENOTYPES AND WHEN WE INTEGRATE 1367 00:49:41,128 --> 00:49:46,033 THE DATA WE HOPE IT CAN -- USING 1368 00:49:46,033 --> 00:49:47,201 COMPLIMENTARY APPROACHES AND CAN 1369 00:49:47,201 --> 00:49:53,741 REALLY TALKING ABOUT MECHANISM 1370 00:49:53,741 --> 00:49:54,575 AND MONITOR. 1371 00:49:54,575 --> 00:49:56,444 SO MY LAST SLIDE IS MOST 1372 00:49:56,444 --> 00:49:56,877 IMPORTANT. 1373 00:49:56,877 --> 00:49:58,612 I COULDN'T DO THIS WORK BY 1374 00:49:58,612 --> 00:50:00,247 MYSELF, I AM LUCKY THAT THE LAB 1375 00:50:00,247 --> 00:50:01,682 HAS ONLY BEEN ACTIVE 1 AND HALF 1376 00:50:01,682 --> 00:50:03,551 YEARS AND I HAVE A REALLY 1377 00:50:03,551 --> 00:50:05,820 FANTASTIC TEAM OF PEOPLE THAT 1378 00:50:05,820 --> 00:50:07,888 WORK WITH ME AND WE LIKE PROTEIN 1379 00:50:07,888 --> 00:50:10,858 NETWORKS THAT WE WORK A LOT OF 1380 00:50:10,858 --> 00:50:12,593 NETWORK BECAUSE WE DO A LOT OF 1381 00:50:12,593 --> 00:50:14,328 APPROACHES AND NEED DIFFERENT 1382 00:50:14,328 --> 00:50:15,029 KNOWLEDGE AND YEAH, I'M VERY 1383 00:50:15,029 --> 00:50:21,335 PROUD OF MY LAB, HOW WELL THEY 1384 00:50:21,335 --> 00:50:22,837 WORK TOGETHER AND ALSO WITH A 1385 00:50:22,837 --> 00:50:24,004 PREVIOUS LAB, WE WORKED TOGETHER 1386 00:50:24,004 --> 00:50:27,608 A LONG TIME AND A NUMBER OF THE 1387 00:50:27,608 --> 00:50:29,043 STUDIES AT THE BEGINNING THAT I 1388 00:50:29,043 --> 00:50:30,578 SHOWED YOU WERE GENERATED, THE 1389 00:50:30,578 --> 00:50:31,879 DATA WAS GENERATED BY A 1390 00:50:31,879 --> 00:50:33,914 FANTASTIC TEAM THAT I WAS 1391 00:50:33,914 --> 00:50:36,383 LEADING IN THE LAB AND WE HAVE 1392 00:50:36,383 --> 00:50:37,685 COLLABORATIONS WITH THE UCSF, 1393 00:50:37,685 --> 00:50:40,855 AND THE LAB THAT WE LOVE AND THE 1394 00:50:40,855 --> 00:50:42,690 BIGGER SCREEN THAT WE'RE NOW 1395 00:50:42,690 --> 00:50:50,498 DOING TOGETHER IN COLLABORATION 1396 00:50:50,498 --> 00:50:51,832 WITH THE BIO-X. 1397 00:50:51,832 --> 00:50:52,500 SO WITH THAT THANK YOU VERY MUCH 1398 00:50:52,500 --> 00:50:53,767 AND I AM HAPPY TO ANSWER ANY 1399 00:50:53,767 --> 00:51:01,609 QUESTIONS YOU MAY HAVE. 1400 00:51:01,609 --> 00:51:04,044 >> YES, ALEXANDRA? 1401 00:51:04,044 --> 00:51:11,886 >> I HAVE A QUESTION FOR THE 1402 00:51:11,886 --> 00:51:13,354 [INAUDIBLE QUESTION FROM 1403 00:51:13,354 --> 00:51:15,990 AUDIENCE -- AWAY FROM MIC ] 1404 00:51:15,990 --> 00:51:16,624 >> THAT'S RIGHT. 1405 00:51:16,624 --> 00:51:21,061 SO THAT'S A VERY GOOD POINT, 1406 00:51:21,061 --> 00:51:21,495 RIGHT? 1407 00:51:21,495 --> 00:51:22,363 WE HYPOTHESIZE THIS FROM PRIOR 1408 00:51:22,363 --> 00:51:24,899 KNOWLEDGE AND ALSO WE ARE NOW 1409 00:51:24,899 --> 00:51:26,534 PURIFYING THE RECEPTOR AND MATCH 1410 00:51:26,534 --> 00:51:28,435 THE PHOSPHORYLATION SITES AND 1411 00:51:28,435 --> 00:51:35,442 BACK THIS UP. 1412 00:51:35,442 --> 00:51:35,643 YEAH. 1413 00:51:35,643 --> 00:51:36,510 >> YEAH, ABSOLUTELY. 1414 00:51:36,510 --> 00:51:36,710 YEAH. 1415 00:51:36,710 --> 00:51:38,045 >> NO, NO, THIS IS A GOOD POINT 1416 00:51:38,045 --> 00:51:39,780 AND WE HAVE TRIED SOME INITIAL 1417 00:51:39,780 --> 00:51:41,282 EXPERIMENTS THAT HAVE NOT BEEN 1418 00:51:41,282 --> 00:51:43,417 SO -- JUST IN THE SENSE OF 1419 00:51:43,417 --> 00:51:49,924 GETTING THE RECEPTOR AND THEN WE 1420 00:51:49,924 --> 00:51:52,126 USE PROTEOMICS AND GPC Rs NOOD 1421 00:51:52,126 --> 00:51:54,194 OTHER ENZYMES SO THAT'S WHAT 1422 00:51:54,194 --> 00:51:55,863 WE'RE PLAYING AROUND WITH TO MAP 1423 00:51:55,863 --> 00:51:57,598 THESE SPECIFIC SIDES. 1424 00:51:57,598 --> 00:51:57,865 [LAUGHTER] 1425 00:51:57,865 --> 00:52:00,267 YEAH, THIS IS A VERY GOOD POINT. 1426 00:52:00,267 --> 00:52:01,669 IT'S ALL INDIRECT, RIGHT BUT WE 1427 00:52:01,669 --> 00:52:06,340 HAVE THEM AT THE PHOSPHORYLATION 1428 00:52:06,340 --> 00:52:07,708 SITE. 1429 00:52:07,708 --> 00:52:08,776 COMPELLING. 1430 00:52:08,776 --> 00:52:09,710 1431 00:52:09,710 --> 00:52:10,377 YEAH. 1432 00:52:10,377 --> 00:52:11,045 YEAH. 1433 00:52:11,045 --> 00:52:12,112 YEAH. 1434 00:52:12,112 --> 00:52:15,449 VERY GOOD POINT. 1435 00:52:15,449 --> 00:52:19,920 YEAH. 1436 00:52:19,920 --> 00:52:25,626 >> I HAVE A QUESTION -- 1437 00:52:25,626 --> 00:52:29,430 [SPEAKING AT SAME TIME ] 1438 00:52:29,430 --> 00:52:39,907 [SPEAKING FOREIGN LANGUAGE ] 1439 00:52:40,507 --> 00:52:41,108 >> TWENTY-ONE, YES. 1440 00:52:41,108 --> 00:52:42,376 >> [INAUDIBLE QUESTION FROM 1441 00:52:42,376 --> 00:52:52,853 AUDIENCE -- AWAY FROM MIC ] 1442 00:52:56,824 --> 00:52:59,693 NYEAH, -- 1443 00:52:59,693 --> 00:53:01,695 >> YEAH, THIS IS A BIAS COMPOUND 1444 00:53:01,695 --> 00:53:03,664 THAT IS BIAS FOR G-PROTEIN 1445 00:53:03,664 --> 00:53:05,699 SIGNALING OR IT INDUCES 1446 00:53:05,699 --> 00:53:08,569 G-PROTEIN SIGNALING OR AT LEAST 1447 00:53:08,569 --> 00:53:11,639 PARTIAL FOR SIGNALING BUT IT 1448 00:53:11,639 --> 00:53:17,778 DOESN'T RECRUIT 1449 00:53:17,778 --> 00:53:18,379 >> [INAUDIBLE QUESTION FROM 1450 00:53:18,379 --> 00:53:18,612 DIRECTLY 1451 00:53:18,612 --> 00:53:19,013 INTERNALIZE AND -- 1452 00:53:19,013 --> 00:53:19,647 >> [INAUDIBLE QUESTION FROM 1453 00:53:19,647 --> 00:53:21,048 AUDIENCE -- AWAY FROM MIC ] 1454 00:53:21,048 --> 00:53:22,583 >> T130 IS A 1455 00:53:22,583 --> 00:53:25,619 BIASED LIGAND, IT'S NOT THIS 1 1456 00:53:25,619 --> 00:53:29,790 BUT THEY HAVE SHOWN ANALGESIC 1457 00:53:29,790 --> 00:53:30,491 EFFECT ACTIVATING G-PROTEIN 1458 00:53:30,491 --> 00:53:34,662 SIGNALING BUT THE IDEA WAS TO 1459 00:53:34,662 --> 00:53:36,864 DESIGN THIS BIAS LIGAND THAT 1460 00:53:36,864 --> 00:53:38,699 DON'T RECRUIT ARRESTIN AND DON'T 1461 00:53:38,699 --> 00:53:42,770 INTERNALIZE WITH THE HOPE THAT 1462 00:53:42,770 --> 00:53:44,405 THE ARRESTIN SIGNALING 1463 00:53:44,405 --> 00:53:46,140 ELIMINATING THEN, IF YOU BLOCK 1464 00:53:46,140 --> 00:53:48,008 THIS IT LIMITS ALL THE SIDE 1465 00:53:48,008 --> 00:53:49,977 EFFECTS, THAT'S NOT QUITE TRUE, 1466 00:53:49,977 --> 00:53:50,978 RIGHT? 1467 00:53:50,978 --> 00:54:01,455 BUT IT'S STILL AN ANALGESIC. 1468 00:54:05,426 --> 00:54:05,926 >> [INAUDIBLE QUESTION FROM 1469 00:54:05,926 --> 00:54:06,260 YEAH, MICHAEL? 1470 00:54:06,260 --> 00:54:06,860 >> [INAUDIBLE QUESTION FROM 1471 00:54:06,860 --> 00:54:17,337 AUDIENCE -- AWAY FROM MIC ] 1472 00:54:33,987 --> 00:54:39,960 BASED 1473 00:54:39,960 --> 00:54:41,095 >> GREAT QUESTION. 1474 00:54:41,095 --> 00:54:43,597 PART IS OPIOID RECEPTOR AND NOT 1475 00:54:43,597 --> 00:54:45,299 SURPRISINGLY REVIEW, LIKE YOU DO 1476 00:54:45,299 --> 00:54:46,633 IN TEXT TILES HOW DOES IT LOOK 1477 00:54:46,633 --> 00:54:48,402 IN NEURONS AND THE NEURAL TYPE. 1478 00:54:48,402 --> 00:54:49,770 AND THAT'S WHAT WE'RE STARTING 1479 00:54:49,770 --> 00:54:51,338 TO GET MORE SYSTEMATICALLY BUT 1480 00:54:51,338 --> 00:54:52,740 FOR THIS RECEPTOR WE USE BECAUSE 1481 00:54:52,740 --> 00:54:55,509 WE COULDN'T GROW THE NEURONS AT 1482 00:54:55,509 --> 00:54:58,345 THE TIME WE USE CELLS SUCH AS 1483 00:54:58,345 --> 00:54:59,613 NEUROBLASTOMA CELL LINE AND WE 1484 00:54:59,613 --> 00:55:01,715 ARE ALSO INTERESTED TO SEE HOW 1485 00:55:01,715 --> 00:55:11,692 LIKE, DOES THE RECEPTOR ENGAGE 1486 00:55:11,692 --> 00:55:12,493 SIMILAR PROTEOME, RIGHT? 1487 00:55:12,493 --> 00:55:13,927 AND IT WAS INTERESTING TO SEE 1488 00:55:13,927 --> 00:55:15,529 THAT THE MAJORITY OF INTERACTION 1489 00:55:15,529 --> 00:55:18,599 ARE CONSERVED BUT THERE ARE 1490 00:55:18,599 --> 00:55:20,734 DIFFERENCES. 1491 00:55:20,734 --> 00:55:23,504 FOR EXAMPLE, WE FOUND COM3, AND 1492 00:55:23,504 --> 00:55:26,974 IT'S VALIDATED IN THE LINE, AND 1493 00:55:26,974 --> 00:55:28,142 THE APPROXIMATE INTERACTION 1494 00:55:28,142 --> 00:55:29,543 NETWORK WE FOUND A DIFFERENT 1495 00:55:29,543 --> 00:55:31,779 NEURON, SO SAME FOR SOME OTHERS, 1496 00:55:31,779 --> 00:55:32,112 RIGHT? 1497 00:55:32,112 --> 00:55:33,847 THEY ARE RELATED BUT MAYBE THE 1498 00:55:33,847 --> 00:55:35,382 FRONT END AND THAT WILL BE 1499 00:55:35,382 --> 00:55:37,551 INTERESTING, WE HAVEN'T -- I 1500 00:55:37,551 --> 00:55:38,318 ACTUALLY HADN'T THOUGHT ABOUT 1501 00:55:38,318 --> 00:55:40,220 THAT BUT I THINK THIS WILL BE 1502 00:55:40,220 --> 00:55:43,257 INTERESTING, RIGHT, WHEN WE NOW 1503 00:55:43,257 --> 00:55:45,225 DO THE SCREEN ACROSS THESE 1504 00:55:45,225 --> 00:55:47,494 DIFFERENT GPC Rs TO LOOK, TO 1505 00:55:47,494 --> 00:55:49,029 LOOK BASED ON EXPRESSION DATA 1506 00:55:49,029 --> 00:55:51,665 RIGHT, ON DIFFERENT CELL TYPES 1507 00:55:51,665 --> 00:55:59,673 CAN IT CAN CONSERVED ON IT. 1508 00:55:59,673 --> 00:56:00,107 >> YEAH. 1509 00:56:00,107 --> 00:56:00,374 >> YEAH. 1510 00:56:00,374 --> 00:56:01,175 >> YEAH, I THINK THAT WOULD BE 1511 00:56:01,175 --> 00:56:02,242 INTERESTING AND I THINK 1512 00:56:02,242 --> 00:56:03,777 SPECIFICALLY WHEN WE -- IF WE 1513 00:56:03,777 --> 00:56:05,179 INITIALLY TART TO HAVE THESE 1514 00:56:05,179 --> 00:56:06,580 KIND OF DATA SETS AND SEE HOW 1515 00:56:06,580 --> 00:56:07,748 DIFFERENT THEY LOOK AND 1516 00:56:07,748 --> 00:56:09,783 DIFFERENT CELLS THEN WE CAN TEST 1517 00:56:09,783 --> 00:56:13,220 THIS IS IF YOU FIND THE CELLS 1518 00:56:13,220 --> 00:56:15,189 ENHANCED DIFFERENT EXPRESSION OF 1519 00:56:15,189 --> 00:56:16,590 THESE NETWORK COMPONENTS, DOES 1520 00:56:16,590 --> 00:56:17,357 IT LOOK DIFFERENT. 1521 00:56:17,357 --> 00:56:18,859 GREAT IDEA. 1522 00:56:18,859 --> 00:56:20,794 I WILL HOPEFULLY IN THE FUTURE 1523 00:56:20,794 --> 00:56:24,431 WE WILL FOLLOW UP ON THIS IDEA. 1524 00:56:24,431 --> 00:56:26,667 I LIKE IT. 1525 00:56:26,667 --> 00:56:27,434 YES, PLEASE. 1526 00:56:27,434 --> 00:56:28,602 >> REALLY WONDERFUL TALK, I HAVE 1527 00:56:28,602 --> 00:56:31,672 A QUESTION ABOUT THE APPROXIMATE 1528 00:56:31,672 --> 00:56:32,906 LABELING. 1529 00:56:32,906 --> 00:56:36,443 IF MY MEMORY'S CORRECT, APEX IS, 1530 00:56:36,443 --> 00:56:38,612 SIZE OF THE APEX IS QUITE BIG SO 1531 00:56:38,612 --> 00:56:40,914 IS THERE ANY EFFECT AT THE 1532 00:56:40,914 --> 00:56:47,354 LABELING OF THE APEX, YEAH, FOR 1533 00:56:47,354 --> 00:56:50,157 I BIOLOGICAL CHARACTERISTIC 1534 00:56:50,157 --> 00:56:50,390 CHANGES? 1535 00:56:50,390 --> 00:56:56,196 >> THE SO THE APEX I THINK IS 1536 00:56:56,196 --> 00:56:58,498 35, I'M NOT SURE THE NUMBER 1537 00:56:58,498 --> 00:57:00,334 CORRECTLY IN MY MIND, THAT CAN 1538 00:57:00,334 --> 00:57:02,169 AFFECT THE FUNCTION IF YOU 1539 00:57:02,169 --> 00:57:04,972 ATTACK A RECEPTOR WITH A LARGER 1540 00:57:04,972 --> 00:57:05,205 PROTEIN. 1541 00:57:05,205 --> 00:57:10,811 SO THERE ARE FEW WAYS HOW WE GO 1542 00:57:10,811 --> 00:57:11,612 ABOUT THIS. 1543 00:57:11,612 --> 00:57:14,114 THE INITIAL STEP IS WE USE A 1544 00:57:14,114 --> 00:57:15,382 FLEXIBLE LINKER WITH THE HOPE 1545 00:57:15,382 --> 00:57:17,451 RIGHT THAT THIS APEX CAN MOVE 1546 00:57:17,451 --> 00:57:20,120 AROUND AND MAKE SPACE FOR 1547 00:57:20,120 --> 00:57:22,289 INTERACTING PROTEIN, AND THEN 1548 00:57:22,289 --> 00:57:26,994 OBVIOUSLY WE TASK FUNCTIONALITY 1549 00:57:26,994 --> 00:57:29,396 OF THE RECEPTOR AFTER -- FOR 1550 00:57:29,396 --> 00:57:31,531 EXAMPLE, A GOOD APPROXIMATION IS 1551 00:57:31,531 --> 00:57:33,967 WHEN WE COMPARE THE APEX 1552 00:57:33,967 --> 00:57:35,369 RECEPTOR AND A NONATTACK 1553 00:57:35,369 --> 00:57:37,371 RECEPTOR WE CAN LOOK AT THE 1554 00:57:37,371 --> 00:57:38,906 SIGNALING OUTCOME THEY PRODUCE, 1555 00:57:38,906 --> 00:57:40,440 LOOK AT TRAJECTORY, CAN SEE IS 1556 00:57:40,440 --> 00:57:42,509 IT SIMILAR TO THE WILD-TYPE 1557 00:57:42,509 --> 00:57:44,244 RECEPTOR OR DOES IT ACTUAL LYE 1558 00:57:44,244 --> 00:57:45,646 EXPRESSOT CELL SURFACE, RIGHT? 1559 00:57:45,646 --> 00:57:48,148 AND THIS IS FOR APPROXIMATION TO 1560 00:57:48,148 --> 00:57:49,583 SAY, OKAY, THE FUNCTION OF THE 1561 00:57:49,583 --> 00:57:51,451 RECEPTOR MIGHT NOT BE IMPAIRED, 1562 00:57:51,451 --> 00:57:52,786 RIGHT? 1563 00:57:52,786 --> 00:57:57,424 THERE'S NO GUARANTEE, BUT THESE 1564 00:57:57,424 --> 00:58:00,861 ARE THE ASSPECS WE TEST FOR FOR 1565 00:58:00,861 --> 00:58:01,194 FUNCTIONALITY. 1566 00:58:01,194 --> 00:58:03,196 AND WHAT WE SEE IS THE RECEPTOR, 1567 00:58:03,196 --> 00:58:05,732 SO THERE ARE SOME GFCRs THAT 1568 00:58:05,732 --> 00:58:07,501 HAVE THIS PDC BINDINGA THE TAIL 1569 00:58:07,501 --> 00:58:10,904 OF THE RECEPTORS AND THESE CAN 1570 00:58:10,904 --> 00:58:13,473 BIND PDC BINDING PROTEINS THAT 1571 00:58:13,473 --> 00:58:14,541 MEDIATE TRAFFICKING AND WHAT WE 1572 00:58:14,541 --> 00:58:17,077 HAVE SEEN FOR, THE BETA 2 1573 00:58:17,077 --> 00:58:18,245 RECEPTOR IS A CLASSIC EXAMPLE 1574 00:58:18,245 --> 00:58:21,848 WHEN WE USE IF, APEX DIRECTLY TO 1575 00:58:21,848 --> 00:58:23,917 THE C-TERMINAL TAIL, WE SEE THAT 1576 00:58:23,917 --> 00:58:26,353 THERE IS A DIFFERENCE COMPARED 1577 00:58:26,353 --> 00:58:29,423 TO THE NONTAGGED RECEPTOR IN 1578 00:58:29,423 --> 00:58:31,858 TERMS OF TRAFFICKING AND THIS 1579 00:58:31,858 --> 00:58:34,227 WAS AN INITIAL PUBLICATION WHEN 1580 00:58:34,227 --> 00:58:36,964 WE TESTED THE APEX, AND WE 1581 00:58:36,964 --> 00:58:38,532 ALWAYS START WITH THE BETA 1582 00:58:38,532 --> 00:58:44,271 RECEPTOR BECAUSE IT'S A WELL 1583 00:58:44,271 --> 00:58:45,072 CHARACTERIZED GPCR RECEPTOR, AND 1584 00:58:45,072 --> 00:58:46,740 WE PLACE IT INTO THE TAIL, WE 1585 00:58:46,740 --> 00:58:49,810 CHOP THE TAIL APART AND PUT IT 1586 00:58:49,810 --> 00:58:51,745 BETWEEN FUNCTIONAL GROUPS AND 1587 00:58:51,745 --> 00:58:54,448 LEAVE THE PDC DOMAIN FREE, THEN 1588 00:58:54,448 --> 00:58:56,850 IT SHOWS SIMILAR FUNCTION IN THE 1589 00:58:56,850 --> 00:58:58,585 WILD-TYPE RECEPTOR, SO MEANING 1590 00:58:58,585 --> 00:58:59,653 IT'S A CONCERN, HOWEVER, I WANT 1591 00:58:59,653 --> 00:59:01,221 TO SAY WITH THE LONG ANSWER TO 1592 00:59:01,221 --> 00:59:03,023 SAYING YES, THERE IS A CONCERN 1593 00:59:03,023 --> 00:59:05,625 THAT CAN CAN EFFECT 1594 00:59:05,625 --> 00:59:07,260 FUNCTIONALITY AND IT'S IMPORTANT 1595 00:59:07,260 --> 00:59:11,765 TO TEST THIS AND WE'RE TRYING TO 1596 00:59:11,765 --> 00:59:13,734 APPROXIMATE THIS SCIENCE BY 1597 00:59:13,734 --> 00:59:14,434 USING FUNCTIONAL ASSAYS. 1598 00:59:14,434 --> 00:59:22,075 >> THANK YOU. 1599 00:59:22,075 --> 00:59:22,409 >> YES. 1600 00:59:22,409 --> 00:59:25,245 >> -- MIGHT HAVE BEEN G-PROTEIN 1601 00:59:25,245 --> 00:59:28,882 AND I WONDER WHETHER YOU OBSERVE 1602 00:59:28,882 --> 00:59:31,485 SIMILAR PATTERNS WITH YOUR 1603 00:59:31,485 --> 00:59:39,092 RECEPTOR AS OPPOSE TO DOMINANT 1604 00:59:39,092 --> 00:59:44,031 PROTEIN AMONG THOSE RELATED 1605 00:59:44,031 --> 00:59:44,464 RECEPTORS? 1606 00:59:44,464 --> 00:59:44,865 >> MM-HMM. 1607 00:59:44,865 --> 00:59:45,265 THAT IS CORRECT. 1608 00:59:45,265 --> 00:59:50,170 SO WHAT I HAVE TO SAY IS THAT, 1609 00:59:50,170 --> 00:59:51,772 YOU KNOW OUR METHOD IS REALLY 1610 00:59:51,772 --> 00:59:54,741 GOOD TO SEE PROTEINS THAT ARE 1611 00:59:54,741 --> 00:59:57,210 NOT CLOSE IN PROXIMITY OF THE 1612 00:59:57,210 --> 00:59:58,178 RECEPTOR BEFORE ACTIVATION AND 1613 00:59:58,178 --> 00:59:59,946 WHEN THE RECEPTOR IS ACTIVATED 1614 00:59:59,946 --> 01:00:01,415 CAN BE RECRUITED RIGHT? 1615 01:00:01,415 --> 01:00:03,617 BECAUSE THEY'RE CHANGING OF -- 1616 01:00:03,617 --> 01:00:06,119 LIKE PROXIMITY AND THAT'S WHAT 1617 01:00:06,119 --> 01:00:08,021 WE LABELED SO THE G-PROTEINS 1618 01:00:08,021 --> 01:00:09,856 LIKE WE LABEL ALL THE G-PROTEINS 1619 01:00:09,856 --> 01:00:12,159 BUT THEY'RE ALREADY IN CLOSE 1620 01:00:12,159 --> 01:00:14,094 PROXIMITY OF THE RECEPTOR BEFORE 1621 01:00:14,094 --> 01:00:16,296 ACTIVATION, SO WE DON'T SEE, 1622 01:00:16,296 --> 01:00:17,798 LIKE FOR EXAMPLE, WHEN WE 1623 01:00:17,798 --> 01:00:19,099 ACTIVATE THE NEW OPIOID SYSTEM, 1624 01:00:19,099 --> 01:00:20,634 WE DON'T SEE A DIFFERENCE OF 1625 01:00:20,634 --> 01:00:22,602 LABELING IN THE INHIBITOR 1626 01:00:22,602 --> 01:00:25,572 G-PROTEIN BECAUSE WE JUST DON'T 1627 01:00:25,572 --> 01:00:27,774 HAVE -- WE DON'T HAVE THE 1628 01:00:27,774 --> 01:00:30,177 TEMPORAL RESOLUTION BUT ALSO NOT 1629 01:00:30,177 --> 01:00:31,044 THE SPATIAL RESOLUTION, THEY'RE 1630 01:00:31,044 --> 01:00:32,279 ALREADY CLOSE IN THE 1631 01:00:32,279 --> 01:00:34,114 NONACTIVATED SO WHAT WE CAN ONLY 1632 01:00:34,114 --> 01:00:35,382 DEDUCE IS FROM THE DOWN STREAM 1633 01:00:35,382 --> 01:00:38,285 EFFECTS THAT WE SEE, SO FOR 1634 01:00:38,285 --> 01:00:42,155 EXAMPLE, WE FOUND WITH THE NEW 1635 01:00:42,155 --> 01:00:43,423 RECEPTOR, AND SPECIFICALLY, IT'S 1636 01:00:43,423 --> 01:00:44,891 ASSOCIATED WITH -- IT'S ACTUALLY 1637 01:00:44,891 --> 01:00:46,493 NOT INTERACTING WITH THE 1638 01:00:46,493 --> 01:00:49,329 RECEPTOR, IT'S INTERACTING WITH 1639 01:00:49,329 --> 01:00:52,165 THE REDUCED GENOME, AND WE SEE 1640 01:00:52,165 --> 01:00:54,367 FOR MANY GI RECEPTOR WE SEE ALL 1641 01:00:54,367 --> 01:00:55,902 COMING UP SO THIS GOOD. 1642 01:00:55,902 --> 01:00:57,537 SO INDIRECT EFFECTS WE CAN READ 1643 01:00:57,537 --> 01:01:00,841 OUT FOR THE G-PROTEIN SIGNALING 1644 01:01:00,841 --> 01:01:02,142 BUT THE DIRECT PROTEIN WE DON'T 1645 01:01:02,142 --> 01:01:02,309 SEE. 1646 01:01:02,309 --> 01:01:05,145 NOW YOU ASK IF WE CAN SEE 1647 01:01:05,145 --> 01:01:07,114 DIFFERENCES AND IT'S TRUE THAT 1648 01:01:07,114 --> 01:01:10,750 WHEN 1 OF THE RECEPTORS THAT WE 1649 01:01:10,750 --> 01:01:13,053 TESTED ACROSS THE RECEPTORS IS 1650 01:01:13,053 --> 01:01:15,856 KNOWN TO PATHWAY GQ AND GI AND 1651 01:01:15,856 --> 01:01:17,724 YOU COULD SEE THAT WE SEE 1652 01:01:17,724 --> 01:01:21,328 NETWORK COMPONENTS THAT ARE 1653 01:01:21,328 --> 01:01:23,163 RELATED TO CONSTANT ACTIVATION 1654 01:01:23,163 --> 01:01:25,632 AND WE SEE PREDICTORS LIKE EY4 1655 01:01:25,632 --> 01:01:27,467 FOR EXAMPLE, SO THIS SAY MERGE 1656 01:01:27,467 --> 01:01:29,603 BETWEEN GI AND GQ IN TERMS OF 1657 01:01:29,603 --> 01:01:30,770 THE INTERACTION NETWORK. 1658 01:01:30,770 --> 01:01:32,005 SO INDIRECTLY WE CAN MAYBE 1659 01:01:32,005 --> 01:01:34,474 OBSERVE IT BUT YOU KNOW THAT'S A 1660 01:01:34,474 --> 01:01:36,977 SHORT COMING OF THE THE METHOD, 1661 01:01:36,977 --> 01:01:39,746 IT'S NOT THE MAGIC BULLET, 1662 01:01:39,746 --> 01:01:41,515 THINGS THAT ARE CLOAT BY THE 1663 01:01:41,515 --> 01:01:42,349 RECEPTOR ACTIVATION, WE CAN 1664 01:01:42,349 --> 01:01:44,918 LABEL BUT WE CANNOT SEE THE 1665 01:01:44,918 --> 01:01:46,086 DIFFERENCE, BASICALLY WITH MOST 1666 01:01:46,086 --> 01:01:49,723 OF THE RECEPTORS WE CAN LABEL 1667 01:01:49,723 --> 01:01:50,857 ALL GENE PROTEINS BASICALLY. 1668 01:01:50,857 --> 01:01:53,193 >> WOULD YOU CONCLIEWT FROM THIS 1669 01:01:53,193 --> 01:01:54,494 THAT THERE'S PREASSEM MYELIN 1670 01:01:54,494 --> 01:01:56,663 ALREADY OF RECEPTOR PROTEIN OR 1671 01:01:56,663 --> 01:01:59,633 JUST SUCH A HIGH DENSITY OF 1672 01:01:59,633 --> 01:02:00,700 G-PROTEIN IN THE CELL? 1673 01:02:00,700 --> 01:02:02,369 >> I THINK THAT'S HARD TO 1674 01:02:02,369 --> 01:02:03,370 CONCLUDE WHAT IT IS. 1675 01:02:03,370 --> 01:02:04,538 I WOULD SAY THEY'RE JUST CLOSE 1676 01:02:04,538 --> 01:02:07,407 BY AND IF THEY ARE PREEXAMINED 1677 01:02:07,407 --> 01:02:12,045 OR NOT, IT'S JUST NOT -- YOU 1678 01:02:12,045 --> 01:02:14,247 KNOW IT DOESN'T HAVE THE 1679 01:02:14,247 --> 01:02:14,714 RESOLUTION TO TELL. 1680 01:02:14,714 --> 01:02:17,050 SO IT'S HARD TO -- I MEAN I WISH 1681 01:02:17,050 --> 01:02:19,486 WE COULD SAY THAT BUT IT'S NOT 1682 01:02:19,486 --> 01:02:20,887 THE MAGIC BULLET THAT ANSWERS 1683 01:02:20,887 --> 01:02:22,455 ALL THE QUESTIONS, YEAH? 1684 01:02:22,455 --> 01:02:32,966 AND THERE'S LIMITATION, RIGHT? 1685 01:02:33,867 --> 01:02:35,035 THANK YOU SO MUCH. 1686 01:02:35,035 --> 01:02:36,503 IF THERE ARE NO FURTHER 1687 01:02:36,503 --> 01:02:40,273 QUESTIONS, THANKS FOR THE GOOD 1688 01:02:40,273 --> 01:02:42,242 DISCUSSION AND THANK YOU FOR 1689 01:02:42,242 --> 01:02:52,385 COMING.