WELCOME TO THE PROTEOMICS
INTEREST GROUP LECTURE SERIES.
TODAY IS MY PLEASURE TO
INTRODUCE OUR FIRST SPEAKER OF
THE REGION 5 DR. RUTH
HUTTENHAIN, SHE WORKS WITH
COLLEAGUES WHERE SHE DID HER
POST DOC AT THE UNIVERSITY OF
CALIFORNIA SAN FRANCISCO WHERE
HE STUDIED PROTEIN SIGNALING
THAT WORKS IN HIV INFECTION.
AND SHE PIONEERED A DRUG
INTERACTION WITH SPATIAL
RESOLUTION AND AFTER HER POST
DOC SHE CONTINUED ADJUNCT
ASSISTANT PROFESSOR AT UCSF AND
RIGHT NOW SHE'S AT STANFORD AND
SHE IS GOING TO TELL US ABOUT
THE RESEARCH THERE AND I WANTED
TO MENTION THAT THIS WAS MADE
POSSIBLE BY ASMS GRANT FOR
GENERAL DISCUSSION, MOST
DISCUSSION GROUPS TO INVITE FOR
THE DISCUSSION.
THANK YOU.
>> WOW, THANK YOU SO MUCH.
SO BEFORE I DIG INTO THE SCIENCE
AND TALK ABOUT PROXIMITY LABELS,
PROTEOMICS AND GPR SIGNALING
FIRST OF ALL I WANT TO THANK
ALEX ANDROGENNIA AND JAN FOR
HAVING ME HERE AND YOU FOR
SHOWING UP AND GOING THROUGH THE
COLD WEATHER.
FROM CALIFORNIA COMING HERE, I
WAS WORRIED ABOUT THE
TEMPERATURE, I HAVE SEVERAL
LAYERS BUT SO FAR I MANAGED
QUITE WELL.
AND WHAT I WANT TO MENTION IS
ALSO IS IT'S A PLEASURE TO BE AT
NIH AND IT'S ALMOST EXACTLY 20
YEARS AGO THAT I WAS HERE WITH
MY FIRST PROTEOMIC EXPERIMENT
AND I ACTUALLY SOME SOME PHOTOS
FROM A DIFFERENT TIME.
SO THAT WAS HERE, 20 YEARS AGO
IN THE BUILDING, THAT'S ME, YES,
20 YEARS, SO THERE ARE SOME
CHANGES THAT HAPPEN IN 20 YEARS
AND IT WAS IN THE BUILDING
ACROSS THE STREET AND THE
BASEMENT AT THE TIME SONIA WAS
LEADING THE MASS SPEC. FACILITY
THERE SO I WAS THERE DURING 3
MONTHS DURING MY MASTERS PROJECT
AND THAT'S WHERE I STARTED MY
PASSION ABOUT PROTEOMICS AND
THAT WAS 20 YEARS AGO AND NEVER
STOPPED AND STILL CONTINUES.
TECHNOLOGY CHANGES, I HAVE
CHANGED, AND FOR ME, --I DON'T
KNOW ABOUT TECHNOLOGY.
LET'S TALK ABOUT THE SCIENCE.
IN GENERAL, I'M INTERESTED IN
CELLS AND I SEE THE CELL AS A
KIND OF COMPUTER WHICH RECEIVES
A LOT OF SIGNALS FROM THE EXTRA
CELLULAR ENVIRONMENT AND THEN
SENSES THEM THROUGH RECEPTORS ON
THE CELL SURFACE AND THEN THIS
INFORMATION IS ON INSIDE THE
CELL THROUGH BIOCHEMICAL
INTERACTIONS THAT ARE MEDIATED
BY THE PROTEINS AND AT THE END
THERE'S CELLULAR RECEPTOR AS A
RESPONSE OF THE CELL.
SO OBVIOUSLY THIS IS NOT LIKE AN
ON/OFF SWITCH, IT IS A
COMPUTATION IN ITSELF AND RATHER
IT'S HAPPENING IN THE SIGNALING
NETWORKS AND IT'S A PROTEIN BUT
I WOULD ARGUE THAT THIS IS
DEFINED BY PROTEIN-PROTEIN
INTERACTION AND HOW THEY ARE
ORGANIZED IN TIME IS SPACE.
SO NOW I'M SPECIFICALLY
INTERESTED IN 1 CLASS OF
G-PROTEIN RECEPTORS AND JUST TO
GIVE YOU A PITCH WHY I THINK
THESE ARE INTERESTING AND
PROBABLY RPGHTS CAN AGREE TO
THAT IS ON THE 1 SIDE THEY
REPRESENT THE LARGEST FAMILY OF
SIGNALING RECEPTORS, SO OVER 800
OF THEM DIVERSE RECEPTORS ARE
EXPRESSED IN OUR BODIES, ALL
OVER, SO PHYSIOLOGICALLY THEY
ARE HIGHLY RELEVANT BUT ON THE
OTHER HAND THERE IS ALSO THE
BIGGEST CLASS OF DRUG TARGETS SO
OVER ONE-THIRD OF THE CURRENTLY
APPROVED DRUGS, WE SEE HERE ARE
TARGETING GPCR, BUT WHEN YOU
LOOK AT THEM IN CLINICAL TRIALS
AS SHOWN HERE FOR VARIOUS
INDICATIONS, GPC Rs ARE THE
DRUG TARGETS.
SO UNDERSTANDING HOW THEY WORK
IS QUITE IMPORTANT
PHYSIOLOGICALLY.
SO A BIG QUESTION WE ARE ASKING
IN THE LAB IS HOW ARE WE
TRACKING THE CELL ACROSS TIME
AND SPACE.
WHY DO WE DO THAT IN IF WE THINK
ABOUT A BIG CLASS OF RECEPTORS
THEY RESPOND TO DIFFERENT
CHEMICALS, COMPOUNDS AND THERE
ARE SOME EXAMPLES, IT'S LIKE A
WIDE PALLET OF DIFFERENT
SIGNALS, AND THEN THERE'S VERY
DIVERSE CELLULAR AND
PHYSIOLOGICAL RESPONSES THAT ARE
KIND OF ARCH CHESTERATED THROUGH
THE ACTIVATION OF RECEPTORS, SO
THE QUESTION IS HOW DOES THE
CELL COMPUTE ALL OF THIS, YEAH,
INSIDE THE CELL.
SO WHAT DO WE KNOW ABOUT GPCR
RIGHT AND IT'S A SIMPLIFIED
DIAGRAM HERE.
SO FIRST WHEN YOU ACTIVATE THESE
RECEPTORS, THEY ENGAGE THE
HETEROTRI MERRIC G-PROTEIN AND
IT CAN BE DIFFERENT FAMILIES OF
THIS SYSTEM, GL5, THEY
ACTIVATION DOWN STREAM PROCESSES
AND MODULATE SECOND RESPONSES SO
THE SIGNALING, THE G-PROTEIN
SIGNALING IS TERMINATED BY
PHOSPHORYLATION OF THE RECEPTORS
THROUGH GRK, SO G-PROTEIN
RECEPTOR KINASES WHICH IS THEN
THOUGHT TO RECRUIT PROTEINS
CALLED BETA ARRESTED THAT
INITIATE INTERNALIZATION TO THE
RECEPTOR TRAFFICKING TO THE
COMPARTMENT SUCH AS ENDOSOMES,
WHERE THEY SIGNAL TO THE
COMPORTMENT AND THEN SIGNAL BACK
TO THE BRAIN, AND IF YOU THINK
ABOUT IT, THEY CAN BE MULTIPLE
GPCR AND EXPRESSED IN THE SAME
CELL, THEY ARE KNOWN TO GI, BUT
TOWARD ANY OF THE PROTEIN, BUT
THEY DO TOTALLY DIFFERENT
THINGS, RIGHT?
THE CELLS RESPOND DIFFERENTLY TO
THE ACTIVATION SO WHEN YOU THINK
ABOUT THIS, IT BECOMES CLEAR
THERE MUST BE MORE
SPECIALIZATION, ORGANIZATION ON
A CELLULAR LEVEL THAT WE
CURRENTLY DON'T UNDERSTAND.
SO THAT'S THE VISION FOR MY LAB,
BASICALLY IS TO UNCOVER THE
BIOLOGICAL MECHANISM BEHIND THIS
DIVERSE ORGANISM AND RESPONSE,
SCIENCE IS A BIG GOAL BUT WHEN
YOU START A LAB, YOU NEED BIG
EXPERIMENTS AND FINDING SOME
INTRS.
SO IF WE HAVE DIFFERENT
RECEPTORS THAT ARE ACTIVATED OR
DIFFERENT RECEPTORS THAT ARE
ACTIVATED WITH LIGAND THEY CAN
LEAD TO DIFFERENT IN VIVO AND
PHENOTYPES AND HOW IS THIS
POSSIBLE?
SO WE WANT TO FILL THIS SPACE
AND THAT'S MY IDEA TO FILL THIS
SPACE WITH MORE, TRY TO REALLY
HAVE A MULTIDIMENSIONAL
CHARACTERIZATION OF THE
SIGNALING NETWORK THAT ARE
ACTIVATED BY THE RECEPTORS BASED
ON, YOU KNOW THE INTERACTIONS
ABOUT THE REFORMS ABOUT,
ACTIVATIONOT SIGNALING THAT ARE
INITIATED AND WHERE THIS IS
HAPPENING AND HOPEFULLY, RIGHT?
THIS IS VERY SIMPLIFIED, THE
EXPERIMENTS DATA NEVER LOOKED
THAT GOOD BUT THE IDEA CAN BE
THEN CORRELATED MOLECULAR EVENT
WITH THE PHENOTYPES THAT WE CAN
READ OUT AND THEN ON THE 1 HAND
GET SOME IDEA ABOUT BIOLOGICAL
MECHANISM, BUT OBVIOUSLY IN THE
LONG RUN THIS HOPEFULLY INFORMED
OF GPCR SIGNAL.
SO THIS IS THE BIG VISION I WORK
ON FOR MANY YEARS BUT WE HAVE
MADE SMALL STEPS TOWARDS IT, AND
THE FIRST THING WE NEED TO HAVE
IS A METHOD THAT CAN TRACK THESE
INTERACTIONS ON SIGNALING EVENTS
THAT ARE ACTIVATED BY THE
RECEPTORS.
SO MY POST DOC SEVERAL YEARS
BACK, WE DEVELOPED A METHOD
WHERE WE USED AN ENZYME CALLED
APEX, THE LABELING ENZYME AND
ENGINEERED PEROXIDASE WHICH WE
FUSE TO THE RECEPTOR.
SOLET ADVANTAGE OF APEX IS WHEN
YOU TREAT THE CELLS WITH BIOTIN
ISSUES AND TO GIVE A VERY SHORT
PAUSE OF HYDROGEN PEROXIDE AND
THEN BIOTIN CAN ALWAYS TURN INTO
RADICAL SAYS, IT'S HIGHLY
REACTIVE AND IT'S LABELED IF A
BIOTIN IS LAID PROTEIN IS IN THE
PROXIMITY OF THE RECEPTOR, SO
THIS IS NICE BECAUSE YOU HAVE
LABELED PROTEINS NEXT TO THE
RECEPTOR.
THIS IS THE PROCESS TREATMENT,
CAN YOU DO THIS BETWEEN
30-SECONDS AND 1 MINUTE OR YOU
CAN TAKE REALLY FOR PROTEOMICS,
YOU CAN HAVE A HIGH TEMPORAL
RESOLUTION SO IT TAKES A
SNAPSHOT OF WHAT'S HAPPENING IN
THE PROXIMITY OF THE RECEPTORS.
NO PROBLEM.
OKAY.
NOW WE CAN CONTINUE.
AND SO THE ADVANTAGE IS NOW WHEN
YOU TAKE THESE SNAPSHOTS OVER
THE TIME COURSE OF THE
ACTIVATION, CAN YOU GET THESE
PROXIMATE PROTEIN NETWORKS OF
THE RECEPTOR OVER TIME, SO THEN
THE NEXT STEP IS THEN, THIS
HAPPENS WITHIN THE CELL, RIGHT,
IN THE LIVING CELL, THEN,
BECAUSE HAVE YOU MARKED THESE
PROTEINS IN PROXIMITY, YOU CAN
LICENSE THESE CELLS FOR ENRICH
THEM WITH BIOTIN PROTEINS AND
ENRICH THEM WITH PROTEOMICS AND
WHAT WE'RE SHOWING IF WE USE
THIS,A PROACH IS THAT WE CAN --
IT'S ACTUALLY THESE ARE KIND OF
RICH DATA SETS, RIGHT?
BECAUSE HERE LIBLED WITHIN THE
RADIUS OF 10-20-NANOMETERS OR A
LOT OF -- IN THE CELL, IT'S
CROWDED, A LOT OF THINGS ARE
HAPPENING BUT WHAT WE HAVE SHOWN
IS THAT WE CAN CONVOLUNTEERSUDE
THIS DATA SET AND GET
INFORMATION ABOUT THE LOCATION
OF THE RECEPTOR BY LABELING
LOCATION SPECIFIC MARKERS OVER
THE TIME POINT BUT ALSO THE TIME
RESULT NETWORKS THAT ARE IN
PROXIMITY THAT MIGHT FUNCTION
WITH THE RECEPTOR.
SO WE HAVE PUBLISHED THIS A
WHILE AGO AND WE USED IT SINCE
AND WE THOUGHT IT'S A QUITE
POWERFUL METHOD TO IN AN
UNBIASED WAY TO STUDY THESE
NETWORKS.
SO WHAT WAS THE LIMITATION, ONCE
WE HAD IT WAS LIKE NO WE WANT TO
APPLY THIS TO MANY LIGANDS AND
ACTIVATE THE RECEPTORS, MANY
DIFFERENT RECEPTORS BUT THEN YOU
RUN INTO A PROBLEM, LIKE THAT'S
ACTUALLY QUITE SOME WORK TO
ENRICH THESE PROTEINS AND WE
WANTED TO AUTOMATE IT.
AND SO THIS IS WHAT WE HAVE BEEN
PUT IN PLACE, RIGHT?
IT'S ACTUALLY NOT ROCKET
SCIENCE, BUT WE THOUGHT IT'S
REALLY HELPFUL, IS THAT WE --
THE TIME LIMITING STUFF AT THAT
TIME WAS THE ENRICHMENT, YOU
HAVE TO DO A LOT OF WASHING
STUFF, EVERYBODY THAT HAS
PROTEIN ENRICHMENT IS AT THAT
POINT QUITE TIME CONSUMING AND
YOU CAN ONLY DO SO MANY SAMPLES
AND WE HAVE AUTOMATED THIS
PROCESS NOW ON A KINGFISHER
FLEX.
IT'S BASICALLY HAS 96 MAGNETS,
MAGNETIC RODS AND HOW WE CAN DO
THIS ENRICHMENT PROCESS AND
WASHING STEP ALL AUTOMATED.
THIS HAS BEEN REALLY NICE AND I
SHOW YOU THIS, IT'S LIKE YOU CAN
REALLY PLAY, IT'S LIKE NIX
PLATES ON THE KING FISH CASE
AUTOMATICALLY UNTIL WE DO THE
BINDING OF THE BIOTINALATED
PROTEINS AND WASH IT OUT THERE
AND THEN WE HAVE THESE READY FOR
SOLUTION.
SO WHAT HELPS IS TO GET A HIGHER
THROUGH PUT RIGHT BECAUSE WE CAN
DO 96 AT THE SAME TIME AND THEN
WE GET LOWER VARIABILITY BUT WE
ALSO USE LOWER INPUT, WHICH ARE
HARD TO DEMAND.
SO JUST TO GIVE A BRIEF LIKE A
LITTLE BIT ABOUT THE TECHNICAL
PERFORMANCE IF YOU COMPARE THE
MANUEL AND AUTOPLATE IT.
IT'S LIKE INVASIVE PROTEIN
IDENTIFICATION, WE SIMPLY
COMPARE THE REPLICATE FROM THE
SAME SAMPLE HERE, AND IT TERMS
OF IDENTIFICATION, RIGHT, THE
MASS SPEC PERFORMS PRETTY
WELL, THE DIFFERENCES ARE NOT
BETTER BUT NOT THAT MUCH, BUT
THEN WHEN YOU LOOK AT HERE THE
QUANTIFICATION, THE CO EFFICIENT
OF VARIATION THAT THEY PLOT HERE
ACROSS ALL PROTEINS IS THAT WHEN
WE AUTOMATED IT, RIGHT, THE CO
EFFICIENT VARIATION IS MUCH
LOWER.
SO WITH THAT, WE CAN DO THIS IN
A MUCH SHORTER TIME AND A HIGHER
REPRODUCIBILITY.
SO WE WANT TO OF COURSE GET
APPROVAL.
CONCEPT RIGHT THAT WE CAN USE
THIS AUTOMATED APPROACH COMBINED
WITH DATA INDEPENDENT
ACQUISITION FOR EVERYBODY THAT
DOES PROTEIN COMPLEX OHMICS, WE
LIKE IT BECAUSE IT'S ALWAYS
REPRODUCED IN THE QUANTIFICATION
AND NOW WE WANT TO SHOW PROOF OF
CONCEPT.
CAN WE USE THIS TO MAP
INTERACTION OF NETWORK OF GPC
Rs AND 1 RECEPTOR THAT WE
TARTED OFF WITH IS THE SEROTONIN
T2A RECEPTOR AND YOU SEE AGAIN
IN THE WORK FLOW, WE FUSE THE
APEX ENZYME TO THE C-TERMINUS OF
THE RECEPTOR IN THE CELL AND WE
HAVE THE T-CELLS, RIGHT, WE CAN
ARGUE IT'S NOT ALL THE RECEPTORS
AROUND THAT CELL LINE BUT IT'S
AT THE BEGENERATEDDING WHEN YOU
ESTABLISH THE METHOD, IS
STRAIGHT FORWARD TO USE AND SO
WE THEN ACTIVATE THE RECEPTOR,
PERFORM THE PROXIMITY LAYER AT
DIFFERENT TIME POINTS OF OVER
THE TIME COURSE OF 30 MINUTES
AND WE THEN, INNOVATOR TIME
POINTS WE INTEGRATE INTO THESE
TIME COURSE STATISTICS.
SO WHAT WE CAN SEE THEN, IF YOU
MODEL THE TIME COURSE DATA, THEN
WE -- ENCLUSTERRED THE CHANGES
IN THE PROXIMITY OF THE
RECEPTOR, WE CLUSTER THEM INTO
THE TRANSIENT EVENT BECAUSE IT'S
HAPPENED, THIS INTERACTION EARLY
AFTER RECEPTOR ACTIVATION AND
THE MORE STABLER LATER
INTERACTIONS THAT COME UP AT
LATER TIMES IN TERMS OF
ACTIVATION.
AND JUST TO ZOOM INTO THIS
TRANSIENT CLUSTER HERE IN THE
BEGINNING, I MAPPED ALL THE
PROTEINS FOR THE 4 CHANGES ON
THE STRING INTERACTION, NETWORK
AND WE ARE QUITE SAFE TO SEE
RIGHT THERE, THERE ARE A NUMBER
OF -- YOU SEE THEM COLOR CODED,
LOOKING AT THE MODEL CITIZEN
WILL KIEWL FUNCTION, THE 1S THAT
ARE INDICATE INDEED BLUE ARE
THINGS THAT HAVE BEEN REPORTED
WE KNOW IN CONNECTION WITH GPCR
SIGNALING AND FOR EXAMPLE, HERE,
WE SEE THE ARRESTIN WHICH WAS I
TALKING BEFORE, AS WELL AS GPC
R14 KINASES, SO THIS IS QUITE
NICE TO SEE THAT WE CAN NOW USE
THIS TO MAP THE INTERACTION OF
THE GFCRs.
SO AS A SUMMARY, RIGHT, WE HAVE
NOW THIS GPCR APEX, PROXIMITY
LABELING METHOD THAT,A LOWS US
TO MAP GPCS TEMPORAL RESOLUTION
AND WE CAN DO THIS ACROSS
MULTIPLE CONDITIONS BECAUSE WE
HAVE MULTIPLE MAPPING.
SO THAT'S THE INPUT AND NOW I
WANT TO TALK ABOUT 2
APPLICATIONS.
THE FIRST 1 IS PUBLISHED
ALREADY, BUT THE SECOND -- SORRY
-- MULTIPLE PIECES THAT ARE NOT
PUBLISHED YET BUT I HOPE IT'S AN
INTERESTING DIRECTION WHERE WE
ARE HEADING NOW.
SO IN THE FIRST PART I WANT TO
TALK ABOUT WHERE WE USE THIS
PROXIMITY LABELING METHOD TO
STUDY THE EFFECTS ON THE
MU-OPIOID RECEPTOR, AS MOST OF
YOU KNOW ISSUES IT'S FOR PAIN
MEDICATION AND BECAUSE OF THE
OPIOID CRISIS, THERE IS A LOT OF
STUDIES AND MEDICATIONS THAT ARE
LESS ADDICTIVE AND SIDE CENTER
FOR CENTER
FOR EFFECTS
AND THERE'S AN INTEREST IN
TRYING TO UNDERSTAND IF YOU HAVE
ANY DIFFERENT LIGAND FOR THE
RECEPTOR, HAVE 1 OF THE
DIFFERENT REGULATORY MECHANISMS
AND SO THIS -- THIS IS MORE
PROOF OF CONCEPT STUDY, THIS IS
ALSO HAS IMPLICATIONS, AND IF IT
CAN EXPAND THIS TO A LARGE
NUMBER OF LIGANDS.
SO NOW WE CHOSE HERE AS A
STARTING -- AS A STARTING SAID,
3 DIFFERENT LIGANDS THAT ARE
CHEMICALLY QUITE DIVERSE, SO WE
CHOSE ANOTHER LIGAND, AND WE
CHOSE THE PZM21 IS A NEW LIGAND
OF CHEMISTRY THAT WAS DEVELOPED
A FEW YEARS BACK.
AND SO WE'RE ASKING THE QUESTION
IF WE USE THE PROXIMITY LABELING
APPROACH OVER A TIME COURSE OF
60 MINUTES HOW DIFFERENT DO
THESE INTERACTION NETWORKS LOOK
LIKE WITHOUT PROTEOMIC METHOD.
AND ALSO, ON THE DATA RIGHT, I
TOLD YOU BEFORE THAT WE -- WE
CAN MAP THE DIFFERENCES IN THE
-- SORRY, THAT WE CAN MAP
DIFFERENCES IN THE CELLULAR
LOCATION OF THE RECEPTOR FAITHS
BAGS OFFICE OF DIVERSITY
PROTEINS AND PROXIMITY BUT ALSO
THE INTERACTION NETWORK AND I
WANT TO SHOW YOU 2 PIECES OF THE
DATA NOW, SUMMARIZE THE STUDY,
SO THE WORN PIECE OF THE STUDY
IS THAT I WANT TO SHOW YOU THE
DIFFERENCE IN CELLULAR LOCATION
THAT WILL PREDICT 1 FROM THE
PROTEOMICS DATA AND THAT'S SHOWN
HERE, SO WE HAVE HERE THE
DIFFERENT CAUSE AND LOCATION,
THEY INDICATE LOCATION ON THE
RECEPTOR THAT WE PROTECT DICT ON
THE MOTE I DON'T MEANIC DATA AND
WHAT WE CAN SEE HERE AND I FIND
REMARKABLE IS THAT BASED ON THE
PROTEOMIC DATA WE CAN REALLY SEE
HOW THE RECEPTOR MOVED THROUGH
THE CELLS, SO WHEN THE RECEPTORS
ACTIVATED WITH THEM, YOU SEE THE
MAIORITY OF THE RECEPTOR, AT
LEAST AS THE PLASMA MEMBRANE,
AND THEN THE PLASMA MEMBRANE
LOCATION DECREASES OVER TIME,
THE RECEPTOR IS INTERNALIZED AND
RELOCALLIZED AND PART OF IT TO
THE LYSOSOME, NOW WE'RE
ACTIVATED WITH MORPHINE, IT'S
KNOWN TO BE A PARTIAL AGONIST,
SO IT DOESN'T REACH THE SAME
ACTIVITY THAN DAMGO, SO NOT AS
MUCH OF THE RECEPTOR IS
INTERNALIZED, AND WITH PZM21, IT
LOOKS VERY DIFFERENT, THE
RECEPTOR STAYSA THE PLASMA
MEMBRANE.
AND WE CAN GET THIS FROM THE
PROTEOMIC DATA.
BUT NOW THE QUESTION IS HOW IS
THE DIFFERENCE IN THE
LOCALIZATION?
HOW IS THE DIFFERENCE IN THE
INTERACTIVE NETWORKS THAT ARE
ASSOCIATED BY THE RECEPTOR,
RIGHT?
AND I SHOW YOU HERE, I SHOW THIS
HERE IN FORM OF THE HEAT MAP, SO
WHAT YOU SEE, THE HEAT OR THE
DIFFERENT CAUSE HERE IS THE
CHANGE IN BIOTINNATION OVER THE
TIME COURSE, THE MORE RED IT IS,
THE STRONGER BIOTINNATION, HAVE
YOU AND I HOPE YOU CAN
APPRECIATE FROM THIS THAT NOT
ONLY THAT'S A BIG DIFFERENCE IN
LOCALIZATION OF THE RECEPTOR,
BUT IT ALSO LOOKED QUITE
DRAMATIC THE DIFFERENCE IN THE
PROTEINS THAT ARE ENGAGED AND
THAT MAKES PROBABLY SENSE RIGHT
BECAUSE OF THAT DEFINITE
TRAFFICKING TRAJECTORY OF THE
RECEPTOR, SO WHAT WE SEE IS THAT
THE BIGGEST EFFECT IN TERMS OF
THE CHANGES IN THE PROTEOME IS
WHEN THE RECEPTOR IS ACTIVATED
WITHIN DAMGO AND THERE ARE
LITTLE EFFECTS WE SEE WITH P21
-- WHEN WE ACTIVATE THE RECEPTOR
OF P21.
WHEN WE PERFORM A SIMPLE GENE
ONTOLOGY ENRICHMENT ANALYSIS TO
UNDERSTAND WHAT MIGHT BE THE
FUNCTION OF ALL THESE PROTEINS
IS THAT WHEN WE ACTIVATE THE
RECEPTOR WITH D AMGO, WE ENRICH
THE PROTEINS THAT ARE MEDIATING
TRANSPORT AND ENDOCYTOSIS, RIGHT
AND THAT DEFINITE LOW MAKES
SENSE AND THERE'S NONE OF THESE
PROTEINS ARE THERE WHEN WE
ACTIVATE THE RECEPTOR WITH P21.
SO WE WENT AHEAD AND WE WANT TO
SAY, WELL, IF WE HAVE THIS DATA
HERE IT CAN BE VALIDATED SOME OF
THESE PROTEINS, ESPECIALLY THE
1S THAT ARE ASSOCIATED WITH
THEM, ARE REALLY REGULATE
RECEPTOR INTERNALIZATION AND
TRAFFICKING, AND SO TO DO THAT,
WE DESIGN -- WE -- OKAY,
SOMEBODY'S NOT ON MUTE.
SO WE DESEEN A FLOW CYTOMETRY
BASED MEASURE TO MEASURE
RECEPTOR TRAFFICKING BUT THAT'S
DIFFICULT BECAUSE THEN WE CAN DO
MULTIPLE PERTURBATIONS AND WE
CAN SEE THE TRAJECTORY.
AND TO DEVELOP THIS, WE WORKED
TOGETHER WITH THE WILLOW COYOTO
LAB AT UCSF, AND WE USED A HELLO
TAG WHICH WE FUSE INTO THE END
TERMINUS OF THE RECEPTOR, SO THE
ADVANTAGE OF THE HALO TAG IS
THAT IT CAN DEFINE DIFFERENT
LIGAND ANDS THEY ARE FLUORESCENT
SO THEY CAN BE FLUORESCENT AND
THEN DIFFERENT CHEMISTRIES ABOUT
THESE LIGANDS SO THEY EXIST IN
CELLS PERMEABLE AND CELL
IMPERMEABLE AND NATURE.
SO WHAT IS NICE NOW IS THAT WE
CAN USE THESE SEQUEPTIALLY.
WE CAN FIRST SATURATE, WE CAN
USE THOSE, AND SATURATE ALL THAT
STUFF IN THE PLASMA MEMBRANE
WITH 1 FLUORESCENT COLOR AND
THEN WE CAN DO A SECOND DAY
LABELING WHERE WE LABEL, WITH
THE PERMEABLE DYE, WE LABEL THE
RECEPTORS AND WE LABEL THEM
INSIDE THE CELL WITH A DIFFERENT
COLOR.
SO THIS IS NEAT, AND WE CAN READ
OUT BY FLOW SIGNIFY TEMETRY,
BASED ON A FLUORESCENT READ OUT
HOW MUCH WAS ON THE CELL SURFACE
AND INSIDE CELL.
AND TO SHOW THAT THERE'S WORK
HERE, HERE'S AN IMAGE WHERE THE
IMAGE FOR THE DIFFERENT CORNERS
WE HAVE HERE, FOR THE RECEPTOR,
AND IT'S AFTER ACTIVATION, SEE
THERE'S' RECEPTOR IN THE CELL
SURFACE, WHICH IS LABELED WITH
THE OTHER WE SEE A SUBSTANTIAL
AMOUNT OF RECEPTOR WITHIN THE
CELL AND WHEN WE CO STAIN THIS
WITH A MARKER THAT'S ALREADY IN
THE ENDOSTUDIES OF MULTIPLE
ENDOCRINE, WE SEE THERE'S A GOOD
COLOCALIZATION, RIGHT?
INDICATING THAT THE RECEPTOR
INSIDE THE CELL IS AT THE
ENDOSOME.
SO THIS LABELED WORK, AND WE CAN
LABEL THE CELL WITH THE
DIFFERENT COLORS AND CAN READ
THIS WITH ACTUALLY CYTOMETRY.
SO WE THEN COMBINE THESE
FLUORESCENT READ OUTS WITH KNOCK
OUTS, AS WE FOUND IN THE
INTERACTION OF THE WORK OF THE
RECEPTOR, SO THIS IS HOW THE
WORK FLOW LOOKS LIKE, WE HAVE
OUR CELLS THAT EXPRESS THE CELL
SET HERE, AND THEN WE KNOCK OUT
THE SELECT CERTAIN FACTORS THAT
I WILL SHOW YOU NOW, IF WE KNOCK
THEM OUT, WE HAVE THE CELLS AND
WE PERFORM THIS ASSAY, WITHIN
AND WITHOUT RECEPTOR ACTIVATION
AND WE CAN MEASURE THE
DIFFERENCE IN CELL SURFACE
RECEPTOR VERSUS INSIDE THE CELL.
AND WE SELECTED FROM OUR
INTERACTION NETWORK, WE SELECTED
8 NETWORK COMPONENTS THAT ARE
PREDOMINANTLY LABELED BIOTIN AND
ACTIVATED WITH DAMGO, WE SHOWED
ARRESTIN AS A POSITIVE CONTROL,
THIS IS INTERNALIZATION, IF WE
KNOCK THIS OUT WE WOULD EXPECT
THERE'S LESS RECEPTOR AND
INTERNALIZED AND MORE ON THE
PLASMA MEMBRANE, OF COURSE WE
INCLUDED NONTARGETTING CONTROLS.
AND JUST TO SHOW YOU HOW THESE
RESULTS LOOK LIKE, I WANT TO
WALK THROUGH THIS HERE, SO
HERE'S FIRST THE NONTARGETTING
CONTROL, WHAT WE PLOT HERE IS
THE NORMALIZED RATIO BETWEEN
RECEPTOR AT THE CELL SURFACE AND
INSIDE THE CELL.
SO THIS IS THAT TIME POINT
THEORIZE THAT WE NORMALIZE THIS
HERE AND WHEN WE ACTIVATE THE
RECEPTOR FOR THE NONTARGETTING
CONTROL, WE SEE THAT SUBSTANTIAL
AMOUNT OF RECEPTOR, THE RACE
CHANGES BECAUSE OF SUBSTANTIAL
RECEPTOR INTERNALIZES.
NOW WHEN WE KNOCK OUT ARRESTIN,
WE CAN SEE THERE'S MUCH LESS OF
THE RECEPTORS INTERNALIZING AND
WE SEE A LITTLE OF THIS BECAUSE
IT'S FOLLICULAR AND KNOCK OUT,
IT'S PROBABLY NOT A HUNDRED
PERCENT KNOCK OUT.
SO THIS IS QUITE NICE.
IT'S A POSITIVE CONTROLS AND WE
WERE WONDERING FOR THE OTHERS,
AND WE CHOSE THAT, HOW THAT
DISTRIBUTION, AND INTERNALIZED
RECEPTOR CHANGES AND WHAT WE SAW
IS THAT FOR 2 PROTEINS I
HIGHLIGHT THEM, AND BPS 35, WE
SEE A SIGNIFICANT DIFFERENCE BUT
IT LOOKED VERY DIFFERENT TO
ARRESTIN, RIGHT?
SO BASED ON THIS DATA, WE
HYPOTHESIZED NOW, THAT COMMD3
AND IT'S MORE RECYCLING OF THE
RECEPTOR BACK TO THE PLASMA
MEMBRANE AND THAT'S WHERE WE SEE
HERE ALREADY AT STEADY STATE
WHERE WE HAVE SOME ALL RIGHT
SOME ACTIVATED RECEPTOR.
WE SEE THERE'S LESS RECEPTOR AT
THE CELL SURFACE AND IN FACT,
THERE'S A RECENT PAPER THAT CAME
OUT THAT CHARACTERIZED MORE
INTENSE 35, IS A PROTEIN PART OF
THE COMPLEX THAT ACTUALLY SHOW
THAT CONTEXT, INVOLVED IN
RECYCLING OF THE NEW OPIOID
RECEPTOR AND THAT MAKES SENSE
WITH OUR FINDING WHICH IS ALWAYS
GOOD TO SEE.
SO JUST TO SUMMARIZE THIS, WE
VALIDATED OTHER PROTEINS WE
FOUND HERE THAT FUNCTION IN
G-PROTEIN SIGNALING BUT I DON'T
HAVE TIME TO GO THROUGH ALL OF
THIS BECAUSE I WANT TO TELL YOU
A BIT ABOUT SOMETHING ELSE,
THAT'S NOT PUBLISHED YET, BUT
JUST TO SUMMARIZE THIS PART, I
THINK WE HAVE, YOU KNOW A
POWERFUL UNBIASED APPROACH FOR
CHARACTERIZING DISTINCT LIGANDS
THAT TARGET GPC Rs AND ALLOWED
US BASICALLY TO LOOKING ACROSS
THE DIFFERENT LIGANDS ALLOWED US
TO DISCOVER ANOTHER REGULATORS
FOR SIGNALING AND TRAFFICKING
AND SO SPECIFICALLY HERE, I
SHOWED YOU THAT WE COULD MAP THE
DIFFERENT TRAFFICKING BEHAVIOR
OF THE RECEPTOR BASED ON THE
DIFFERENT LIGANDS, BUT THEN WE
CHARACTERIZE THEM AT CP35 AS
PROTEINS THAT ARE FUNCTIONING IN
THE RECEPTOR RECYCLING AND WE
FOUND THAT KCTA12 AND 2 PROTEINS
ARE PROTEINS THAT REGULATE
G-PROTEIN STASIS.
SO SO NOW WE WANT TO GO TO THE
NEXT STEP NOW THAT WE HAVE
TESTED IN AND LIGANDS, HOW IS IT
IF WE COMPARE DIFFERENT
RECEPTORS, RIGHT?
HOW SIMILAR ARE DIFFERENT DEC
APPROXIMATE WE CAN MAP WITH OUR
METHOD, SO THAT'S WHAT I WANT TO
TALK ABOUT IN THE SECOND PART
WHERE WE USE THE SAME APPROACH
TO UNCOVER ANOTHER REGULATION OF
GPCR SIGNALING TO ANOTHER
RECEPTOR.
SO TO START THIS UP RIGHT WE
SAID OKAY, LET'S SELECT A NUMBER
OF DIFFERENT RECEPTORS AND THEN
THIS WAS PART OF A LARGER SCALE
PROJECT THAT I STARTED AT UCSF
IN COLLABORATION WITH US THAT
WAS GEARED TOWARD FINDING NOVEL
PAIN MEDICATION, SO THIS IS WHY
THE RECEPTORS WE CHOSE HERE ARE
RELATED SOMEHOW HAVE SOME
FUNCTION IN PAIN REGULATION,
HOWEVER, WHAT I'M TALKING ABOUT
IS GENERAL BIOLOGY OF THE
RECEPTOR, BUT THAT WAS A
SELECTION CRITERIA, SO WE CHOSE
DIFFERENT RECEPTOR FAMILIES
AMONG THEM, THE OPIOID RECEPTOR,
I TALKED ABOUT THE NEW OPh
RECEPTOR BUT THERE ARE MULTIPLE
RECEPTORS IN THIS FAMILY,
RF-AMYDE, PROSTAGALA NDIN
RECEPTORS, AND THEY HAVE KNOWN
PROTEINS, AND THESE HAVE A
DEFINITE LIGAND WE CHOOSE TO
ACTIVATE AND SO WE HAVE
DIFFERENT, DIFFERENT RECEPTOR
FAMILIES AND THEY HAVE DIFFERENT
FOR G-PROTEIN SO HOW THEN WE
WERE WONDERING IF YOU USE OUR
GPCR APEX APPROACH, HOW SIMILAR
ON OTHER NETWORKS THAT THE
RECEPTOR ENGAGES AND CAN WE USE
THESE AGAIN AS WE FIND THINGS
THAT ARE COMMON ACROSS
RECEPTORS, BUT ALSO THINGS THAT
LOOK DIFFERENT, RIGHT?
AND AGAIN I SHOW YOU KIND OF BIG
DATA BUT I DON'T GO THROUGH ALL
OF THIS IN DETAIL SO WHEN WE
LOOK HERE, THESE ARE THE RESULTS
WE GET OUT FROM THE APEX
APPROACH, THIS IS AGAIN A BIG
HEAT MAP THAT DOESN'T SAY SO
MUCH AND IMEAN I DON'T WANT TO
GO THROUGH DETAILS OF ALL OF
THIS, BUT I CAN SAY IS THAT WHEN
WE LOOK ACROSS THE RECEPTORS,
THERE ARE OBVIOUSLY SOME SIMILAR
PATTERNS, RIGHT?
WHICH IS NICE TO SEE AND THEY
MAKE SENSE FOR EXAMPLE, WHEN WE
LOOK AT THE 1S THAT ARE
INDICATED, THESE ARE ALL THE
G-RECEPTORS WHICH LEADS TO AN
INCREASED IN INTRACELLULAR
CALCIUM AND WHEN WOOK AT GENE
ONTOLOGY ENRICHMENT OF THE
PROTEINS IN DIFFERENT CLUSTERS
WE SEE CALCIUM DEPENDENT PKC
ACTIVITY IS ENRICHED FOR
RECEPTORS THAT CAPPA HQ.
WE SEE THIS SMIPGS AT THE
EXPENSE, A LOT OF SIGNAL FOR
ENDOCYTOSIS OR ENDOSOME FOR
RECEPTORS AND WE KNOW UNDERGO
TRAFFICKING LIKE INTERNALIZATION
WHICH ALSO MAKES SENSE, BUT 1
THING IS LIKE WHEN YOU LOOK AT
THIS RIGHT, IT'S NOT ALWAYS
THROUGH T-CELL WHAT YOU FALL OFF
ON BUT 1 THING THAT WAS REALLY
STRIKING TO US IS THAT THIS
MPFS1, WHICH LOOKS ARE HAVE
DIFFERENT THAN OTHER RECEPTORS
HERE IN THIS HEAT MAP.
IT'S A GI COUPLED RECEPTOR,
INHIBITOR G-PROTEIN OPIOID
RECEPTOR BUT THE PROFILE LOOKS
COMPLETELY DIFFERENT IN THE
OTHER RECEPTORS.
AND WE SEE THERE'S SOME
ENRICHMENT THAT IS SPECIFIC FOR
MPP A 1, WHICH IS FOR THE LIGAND
COMPLEX, SO NOW TO SIMPLIFY OUR
RESULTS THERE, I WILL FOCUS JUST
ON MPF 1 AND I WILL FOCUS ONLY
ON THE PROTEINS THAT ARE SILG 95
CANTILY CHANGING IN BIOTINNATION
FOR THIS RECEPTOR.
SO HERE THAT'S MUCH MORE SIMPLE
TO LOOK AT, RIGHT IN WHETHER WE
CAN SEE HERE WHEN WE FOCUS ON
THIS IS THAT THERE'S A FEW
PROTEINS IN STRONG CHANGES IN
BIOTINNATION AND WHEN WE LOOK
INTO THEM DEEPLY IS THAT THESE
ARE -- THESE ARE MEMBERS OF THE
SAME COMPLEX, SO THESE ARE
MEMBERS OF A LIGAND, COLORING E3
LIGANDS SO BTRC, AND THEY ARE
SUBSTRATED THERE OF THIS
COLORING HISTORY LIGAND COMPLEX,
IT MEANS THEY BIND SUBSTRATES
THAT ARE THEN BECOME
UBIQUITINNATED, THE LIGASE ARE
MODULAR, AND THEY ARE HIGHLY
SIMILARITY IN TERMS OF THE
SEQUENCE SPECIFICITY, THEY ARE
BOTH SUBSTRATE WITH THIS.
SO THIS WAS QUITE INTERESTING,
WHEN YOU SEE SOMETHING LIKE
THIS, YOU DON'T -- MULTIPLE
MEMBERS OF THE SAME COMPLEX
COMING UP IN A DATA SET THAT'S
PROBABLY NOT HAPPENING BY CHANCE
ISSUES -
RIGHT?
WE WANT TO VALIDATE IT AND
RETURN IT WITH COMPLIMENTARY
TECHNOLOGIES AND WE WORK CLOSELY
WITH THE LAB AND IMAGING AND
WHAT WE CAN SHOW WITH THEM HERE
IS WHEN WE IMAGE THE -- WHEN WE
IMAGE WE ACTIVATE THE RECEPTOR,
WHEN WE HAVE THE NONACTIVATED
RECEPTOR, THEN SBX W11 IS
COMPUTE IN THE SIDE, WHEN WE
ACTIVATE THE RECEPTOR WITH
NPFFR1, IS THAT THE SUBSTRATE
RECEPTOR REALLY CAN RELOCALLIZE
TO THE PLASMA MEMBRANE AND CO
LEGALAISE WITH THE RECEPTOR --
CO LOCALIZE WITH THE RECEPTOR.
THIS ISN'T IN HEX CELLS BUT IS
THIS SOMETHING CELL TYPE
SPECIFIC, RIGHT?
AND NPFFR1 IS ACTUALLY WHEN IT'S
EXPRESSED IN CELLS THAT HAVE A
PRIMARY ENTHELIUM, IT IS
ENRICHED COMPARED TO THE PLAZ
MIDS IN MEMBRANE.
SO WHEN I SAY WE, THIS IS MARK'S
LAB, WHAT WE COULD SHOW IS THAT
THIS IS 1 THELIUM, AND IT'S REEL
KEEPSAKESLY NICELY AND ENRICHES
THE THELIUM CAN AND NOW WITHOUT
ACTIVATION, FBXW11 IS IN THE
BODY BUT YOU DON'T SEE IT IN THE
THELLIAL CELLSIUM ITSELF AND
WHEN IT'S ACTIVATED YOU SEE IT
ENRICHED HERE AND CO LOCALIZED
WITH THE RECEPTOR SO THIS IS
SOMETHING THAT IS NOT CELL
SPECIFIC WHICH IS GREAT, RIGHT?
SO THE NEXT THOUGHT WE IS, WELL
WE HAVE QUITE KNOWLEDGE ABOUT
THESE E3 LIGASES, RIGHT?
SO LIGASES THEY ARE RECRUITED TO
A PROTEIN TO UBIQUITINNATE THE
PROTEIN ITSELF OR SOMETHING IN
THE SURROUNDING.
SO WHAT WE KNOW FOR THIS
SPECIFIC SUBSTRATE RECEPTOR,
FBXW11 THEY RECOGNIZE THIS
POSSIBLE DIAGRAM WITH THIS
SEQUENCE AND WE WERE QUITE LUCKY
BECAUSE FOR A LOT OF LIGASES
THAT IS NOT THE FORM.
SO WHEN WE LOOKED INTO THE
RECEPTOR STRUCTURE THAT IS HERE
OR THE RECEPTOR SEQUENCE, WE
FOUND IN THE C-TERMINALS HERE,
WE FOUND THIS SEQUENCE WHICH IS
GREAT.
SO FROM THIS FINDING THEN OUR
HYPOTHESIS IS THAT THE RECEPTOR
MIGHT BE PHOSPHORYLATED UPON
ACTIVATION AND OUR GUESS WAS
BECAUSE OF THE THE G-PROTEIN,
THAT'S OUR FIRST GUESS, RIGHT,
THESE ALSO PHOSPHORYLATE NPFFR1
AND THEN THIS PHOSPHORYLATION OF
THIS LEADS TO RECRUITMENT OF
THESE 3 LIGAND COMPLEX, SO NOW
IF ANYONE WANTS TO TEST THIS
HYPOTHIS THERE IS 2 WAYS WE WENT
ABOUT IT TO PERTURB THE SYSTEM.
SO ON 1 HAND WE CAN MUTATE THE
FOSTER NURSED FOCUSED ON
FOSTER
NURSED FOCUSED ON DEGRADATION
RON, AND WE FOUND THAT WITH THE
LIGASE AND ON THE OTHER HAND,
THERE INHIBITORS FOR GRK, AND A
GOOD INHIBITOR FOR GNK, 2 ASK 3,
AND THIS 1 TARGETS SPECIFICALLY
2 ANDLY AND TO BE HONEST WE JUST
TARGET THIS BECAUSE THAT'S GOOD
COMPOUND AVAILABLE, RIGHT?
THIS IS THE WHAT WE DO, YOU HAVE
AVAILABLE TOOL SO WE DID THIS.
AND SOMETIMES BIOLOGY TURNS OUT
TO BE TRUE AND KIND TO US,
RIGHT?
THIS EXPERIMENT, SO WHEN WE NOW
COMPARED THE DIFFERENT
CONDITION, WE PERFORMED THE
APEX EXPERIMENT THEN FOR ACROSS
THIS CONDITION FOR THE WILD-TYPE
RECEPTOR FOR THE MUTANT TNT MA
DOESN'T HAVE THE POSSIBLE DEGRON
ANYMORE, AND ALSO THE 1 TREATED
WITH COMPOUND 101.
IF WE NOW SIMPLY LOOK AT THESE
COMPONENTS OF THE LIGASE, WE SEE
THAT THIS CHANGE IN
BIOTINNATION, WHEN WE ACTIVATE
THE WILD-TYPE, WITH OTHER
CONDITIONS RIGHT, IT'S
COMPLETELY BLOCKED, THE LIGASE
IS NOT RECRUITED TO THE
RECEPTOR.
SO THIS WAS -- THIS WAS GREAT.
SO WE THOUGHT, PROBABLY OUR
HYPOTHESIS IS TRUE, RIGHT?
AND THEN OF COURSE, WE WANTED TO
PROVIDE IT AGAIN WITH A METHOD,
ABOUT YOU WE ALSO THOUGHT, WELL,
IF THIS PHOSPHORYLATION IS
MEDIATE BY GRK, THEN THE
CANNONICLE MODEL THEN MEDIATED
PHOSPHORYLATION LEADS TO
RECRUITMENT OF ARRESTINS, THE
QUESTION S&P THIS INTERACTION
DEPENDENT OR INDEPENDENT OF
ARRESTIN?
SO TO TEST THIS RIGHT THEN WE
WORK WITH THE LAB, SPECIFICALLY
A GRADUATE STUDENT IN THE LAB
AND THEY HAVE THIS 9
COMPLEMENTATION ASSAY CENTER
WHERE WE HAVE 1 PART OF THE
LARGE BIT, WE HAVE TARGET TO THE
PLASMA MEMBRANE, AND THE SMALL
BIT WE CAN TARGET TO ANY
PROTEINS WE ARE INTERESTED IN.
IN OUR CASE, WHAT WE EXPECT IS
IF WE -- IF WE USE THE LIGASE,
IF WE ACTIVATE OUR RECEPTOR THAT
THE FAW11 RELOCALLIZE TO THE
PLASMA MEMBRANE AND THEN WE GET
THE LUMEN SIGNAL.
SO WE NOW USE THIS IN A
DIFFERENT CONDITION, SO WE
AGAIN, WE TARGET THIS MODEL TO
THE 11, WE INCLUDED WILD-TYPE
RECEPTOR, INCLUDED THE MUTANT
RECEPTOR THAT THE SHOW BEFORE
AND A PROTEOMIC DATA THAT
DOESN'T RECRUIT THE E3 LIGASE,
BUT THEN WE ALSO WANTED TO HAVE
THE NEW OPIOID RECEPTOR AS AN
EXAMPLE OF CLASSIC RECEPTOR THEN
WHEN YOU ACTIVATE IT, IT
RECRUITS, IT'S PHOSPHORYLATED BY
GRK AND RECRUITS ARRESTIN.
SO JUST AS A COMPARISON, RIGHT?
AND WHAT WE CAN SHOW HERE OR THE
E3 LIGASE, IS AS SPECIFICALLY
FOR THE WILD-TYPE, IT DOESN'T
HAPPEN FOR THE MUTANT RECEPTOR
BUT IT ALSO DOESN'T HAVE --
WHICH WE WOULD EXPECT.
NOW WE DO THE SAME EXPERIMENT,
WHERE WE TARGET THE SMALL, BUT
TO ARREST IT.
SO WHAT WE SEE HERE IS AN
OPPOSITE PICTURE, AND THE STRONG
LUMINESCE ENSEL SIGNAL, RIGHT?
BECAUSE THE ARRESTIN IS
RECRUITED THROUGH A REPRESENTOR
THROUGH ACTIVATION AND
POSFORALATION THROUGH GRK, BUT
NEITHER FOR THE WILD-TYPE TYPE,
FOR THE MUTANT RECEPTOR WE HAVE
ARRESTIN RECRUITMENT.
SO THIS IS VERY INTERESTING,
RIGHT WHAT WE CONCLUDE SO FAR
FROM THIS, IS THAT WE NOW HAVE,
WE DISCOVER A NOVEL INTERACTION
BETWEEN THE NPFFR1 RECEPTOR AND
THE 3 LIGASE, USING OUR
PROTEOMIC APPROACH AND BEING
LOAMACYING ACROSS DIFFERENT
RECEPTORS AND WHAT IS GREAT IS
THAT WE CAN SHOW THAT THESE
INTERACTIONS MEDIATE BY THE
FOSTER NURSED FOCUSED ON DEGRON,
OF THE TAIL OF THE RECEPTOR
WHICH IS PHOSPHORYLATED THROUGH
GRK AND INDEPENDENT OF THE
RESIDENCE.
OF COURSE NOW, THE BIG QUESTION
IS AND I AM SORRY I CANNOT GIVE
YOU THE ANSWER FOR IT BECAUSE
WE'RE STILL LOOKING AT THIS,
RIGHT, THE QUESTION IS WHAT THE
FUNCTION OF THIS RECRUITMENT,
RIGHT?
AND SO WE ARE LOOKING AT WHAT IS
THE EFFECT ON THE G-DEPENDENT
PROTEIN SIGNALING BUT ALSO
BECAUSE IT'S AN E3 LIGASE, AND
THE RECEPTOR, WHAT IS THE EFFECT
ON RECEPTOR'S ABILITY SO IT'S
SOMETHING THAT CONTINUED IN MY
LAB AND GOOD FOR FOLLOWING UP
ON.
YOU KNOW WHAT?
I STILL HAVE SOMETIME, RIGHT?
I WANT TO SHOW THE LAST PART
THAT IS IN PROGRESS NOW BECAUSE
WHAT WAS -- WHAT BECAME CLEAR TO
US BASED ON THIS EXAMPLE, IS
THAT MAYBE THERE'S MORE TO IT
THAN THIS CANNONICLE MODEL,
RIGHT?
THAT MAKES SENSE, RIGHT?
WHEN YOU THINK ABOUT GPC Rs
THEY HAVE A C-TERMINAL TAIL AND
SOMEHOW ALSO SOME OF THE LOOPS
CAN BE PRETTY LONG.
THEY'RE VERY UNSTRUCTURED,
RIGHT?
YOU HAVE PHOSPHORYLATION OF
THESE AUTOSECRETION, THEY CAN
HAVE CONDITIONING FOR THE
SITUATION WHERE THEY CAN ENGAGE
PROTEINS AND SOMEHOW, THIS SEEMS
VERY SIMPLE TO THINK THERE ARE
MULTIPLE GRKs THAT CAN
PHOSPHORYLATE THE DIFFERENT
RECEPTOR TAILS AND THESE TAILS
CAN BE QUITE DEFINITE IN TERMS
OF LENGTH AND SEQUENCE AND THEY
MIGHT RECRUIT OTHER PROEN TOOS
THAT ARE ONLY ARRESTINS, RIGHT
AND THIS IS NOT ONLY BASED ON
OUR FINDINGS, SO THIS IS THE
CLASSIC MODEL HERE, RIGHT, THE
GRK PHOSPHORYLATOR ARRESTIN AND
THEN THE PHOSPHORYLATION AND
MEDIATION.
BUT THERE'S MUCH MORE EVIDENCE
EMERGE NOTHING LITERATURE THAT
THERE'S GRK REGULATION AND
RECRUITMENT SO WE FOUND THIS
INTERACTION OF THE NPFFR1, AND
INDEPENDENT OF ARRESTIN AND IT'S
BEAUTIFUL WORK BY BEN WHO HAS
SHOWN THAT USING THE RECEPTOR IS
IMPORTANT FOR DEVELOPMENT, WHEN
ACTIVATED IT PHOSPHORYLATES BY
GRK WHICH LEADS NOT TO THE
RECRUITMENT OF ARRESTIN, BUT THE
SUBUNIT OF PKAA, AND I HAVE
SHOWN NICELY THIS IS A KEY EVENT
TO REGULATE TRANSCRIPTION DOWN
STREAM OF FOX.
AND IT'S BASED CONDITIONALLY ON
THE SEQUENCE OF THE C-TERMINUS
TAIL AND THEN THERE ARE A NUMBER
OF STUDIES SHOWN FOR A NUMBER OF
RECEPTORS THAT ARE KNOWN TO
INTERNALIZE AND ARRESTIN IN AN
INDEPENDENT WAY.
SO WE THOUGHT WHILE THIS
EMERGING DATA IN THE LITERATURE,
EXAMPLES, CAN BE MAY BE USE OUR
PROTEOMIC APPROACH TO IN A
LARGER SCALE TO EXPLORE 1 GRK
FUNCTION BEYOND ARRESTIN
RECRUITMENT IN THE SYSTEMATIC
MANNER, AND IT'S ACTUALLY QUITE
AN UNDERTAKING SO I HAD THIS
IDEA, MAYBE SEVERAL MONTHS AGO
AND I THOUGHT IF WE EMBARK ON
THIS, WE HAVE TO DESIGN THIS
REALLY WELL AND WHAT YOU ALSO
NEED IS LIKE A STUDENT WHO IS UP
FOR A BIGGER CHALLENGE.
SO THIS IS SOMETHING THAT WE ARE
GEARING UP TOWARDS NOW AND I
JUST WANT TO SHOW YOU JUST TELL
YOU HOW WE'RE THINKING OF DOING
IT AND BITS AND PIECES OF A
LITTLE BIT OF DATA, NOT TOO MUCH
BUT JUST TO GIVE YOU A SENSE OF
WHAT I HOPE WE CAN FIND FROM
THIS.
SO OUR IDEAS, MAYBE A BIT CRAZY
BUT I THINK THE STRENGTH COMES
IN NUMBERS, SO WE -- WE CHOOSE A
NUMBER OF RECEPTORS, RIGHT IN
AND OUR INITIAL TARGET IS MAYBE
50, AND THE QUESTION IS HOW DO
WE SELECT THEM, RIGHT?
SO WE THOUGHT, WE CAN MAKE THE
SELECTION BASED ON A DIFFERENT
LENGTH AND COMCISION OF
C-TERMINAL TAILS, RIGHT?
THEN OF COURSE, WE SELECT ONLY
RECEPTORS THAT ARE NOT WORKING
SO YOU HAVE NEGATIVE SELECTION,
AND THEN, YOU KNOW WHAT WE
ACTUALLY FOUND QUITE INTERESTING
IS THAT ALPHA FOLD IS NOT GOOD
FOR A NUMBER OF STRUCTURES AND
WE DON'T KNOW HOW GOOD IT IS,
BUT FOR A SPECIFIC EXAMPLE OF
SMOOTHNESS, IT CAN PREDICT
CONDITIONALLY FOR THE STATE.
AND WHAT WAS FOUND IN THE
EXAMPLE OF SMOOTHING WHERE IT
ENGAGES THE PK,A,A, THAN ALPHA
FOLD PREDICT THIS SHORT ALPHA
HELIX THERE WHICH ACTUALLY BINDS
CATALYTIC SUBUNIT OF PKAA AND WE
THOUGHT WE COULD ALSO USE ALPHA
FOLD THEN, TO FIND DIFFERENT
REGIONS ACROSS RECEPTORS,
DIFFERENT REGION OF THE
C-TERMINUS TAIL, AND AT THE END
WE ARE TESTING THIS HYPOTHESIS.
SO THIS IS THE WAY THAT WE
APPROACH THIS.
NOW I CANNOT TELL YOU IF THAT'S
A VARIED APPROACH OR NOT, BUT WE
CHOOSE THEM WHERE WE SEE
INTERESTING ALPHA HELICAL
PREDICTIONS AND SOME WHERE WE
DON'T SEE PREDICTION AND SEE
WHAT COMES OUT OF OUR STUDY.
SO THIS IS HOW WE,A PROACH NOW
THE SELECTION OF THE TCR.
THEN WHAT WE THOUGHT IS INSTEAD
OF JUST DOING THE APEX
EXPERIMENT IN WILD-TYPE CELLS,
WHAT WOULD BE ACTUALLY VERY
INFORMATIVE IF WE DIRECT USE 3
DIFFERENT FLAVORS OF CELL LINES,
WE USE WILD-TYPE CELLS, WE USELE
CELLS THAT HAVE ARRESTINS
COMPLETELY KNOCKED OUT SO THAT
WE CAN SEE OTHER INTERACTIONS
THAT ARE DEPENDENT ON ARRESTINS
AND THEN -- AND THE 1 THAT HAS
GRK KNOCK OUT SO WE CAN SEE
THESE ON GRKs AND I THINK
WHILE THIS TROUBLE THE AMOUNT OF
WORK UPFRONT WE CAN GET A LOT OF
INFORMATION FROM IT UPFRONT AND
I TOLD YOU WE HAVE THE
ENRICHMENT SO WE THOUGHT WE ARE
GEARED SO IT MIGHT BE FEASIBLE,
RIGHT BUT THEN WHEN YOU WANT TO
PUT 50 GPCR IN THE 3 DIFFERENT
CELLS, THAT'S QUITE AN
UNDERTAKING SO WE THOUGHT TO
ALSO FIND AN ELEGANT APPROACH
HOW WE CAN DO THIS IN THE
MOLECULAR BIOLOGY WAY, SO WE
INTEGRATE THESE LANDING PAD INTO
THE 3 CELL LINES AND WHAT I
MEAN, THIS LANDING PAD WHEN WE
CAN EASY SWAP IN AND OUT
RECEPTORS OF THIS AND WE HAVE
BEEN DOING THAT AND MAKING THESE
CELL CHARACTERIZATION.
AND THIS IS WHAT WE HAVE
CURRENTLY SET UP.
SO THE NEXT STEPS ARE THEN OF
COURSE TO DO THE GPCR SELTZMENT
AND AGAIN, WHAT I WAS HOPING IS
THAT THE DATA WOULD LIKE LIKE
THIS A LITTLE BIT.
WE HAVE THEM WHEN WE LOOK AT
ACROSS A DIFFERENT CELL LINE AND
RECEPTORS, WE CAN FIND
INTERACTION THAT IS POST GRK AND
THE ARRESTIN INDEPENDENT OR GRK
DEPENDENT SO BY THIS, RIGHT IN
THIS IS HELPFUL.
BEFORE YOU -- BEFORE YOU EMBARK
ON THIS HUGE STUDY, YOU WANT TO
ACTUALLY SHOW THAT THIS IS
ACTUALLY TRUE FOR SOME -- FOR
PRIOR KNOWLEDGE, SO JUST AS A
SANITY CHECK RIGHT WE DID THIS
FOR NFPPR1, AND WE HAVE THIS
INTERACTION OF THE LIGATES
INDEPENDENT OF ARRESTIN AND WE
DID THE APEX EXPERIMENT ACROSS
THE 3 CELL LINES AND WHAT WAS
REALLY STRIKING TO SEE, I
THOUGHT THIS WAS SO GOOD TO SEE,
YOU DON'T EVEN NEED STATISTICS
FOR THIS, IS REALLY THE E3
LIGASE COMOPPOSITE BEHAVIORIAL
PHENOTYPENTS THAT COMPLETELY
DISAPPEAR IN THE GRK KNOCK OUT
CELL LINE WHEN WE HAVE THE
ARRESTIN KNOCK OUT, THEY LOOK
BASICALLY IDENTICAL, AND THEN WE
SEE ALSO DOWN STREAM
INTERACTION, SO FOR EXAMPLE,
HERE, THIS IS -- THESE ARE ALL
MEMBERS OF THE ESCORT LIKE
COMPLEX, THAT ARE LIKE UBIQUITIN
READ OUTS SO THE HYPOTHESIS FROM
THIS EXPERIMENT, AND YOU SEE
WHEN YOU LOOK AT THE TIME SCALE,
SEQUENT QUALLY THEY COME UP
LATER ABOUT YOU THEY'RE ALSO PDK
DEPENDENT SO OUR HYPOTHESIS IS
THAT THE LIGASE HERE IS
UBIQUITINNATING THE RECEPTOR AND
WE ARE VALIDATING AND I THINK WE
ALSO DID A SIMILAR EXPERIMENT,
AND WE ALSO GOT THERE, GRK
ARRESTIN INDEPENDENT
INTERACTION, LOOKS AT THE
DIFFERENT 1 HERE AND I THINK IF
WE HAVE THIS KIND OF DATA ACROSS
THE RECEPTORS WE CAN ACTUALLY
COME UP WITH REALLY NICE
HYPOTHESIS TO FOLLOW UP BUT WE
CAN MAYBE THEN ALSO, MY HOPE IS
RIGHT, WE CAN GO BACK TO LOOKING
INTO THE SEQUENCES OF THE
C-TERMINALS REPAIR OF THE
RECEPTOR AND FIND SOME SORT OF
PAIR UP, MAYBE.
SO THIS WAS ONGOING, BIG EFFORT
BUT I'M SUPER EXCITED FOR THE
RECEPTOR I'M SO EXCITED TO SEE
THE DAILY BASIS THEA -- DATA AD
SEE IF WE CAN SEE HOW THIS
PANNED OUT.
THE LAST BIT OF DAT A WANT TO
SHOW YOU AND IT'S REALLY ONLY 1
SLIDE, I SEE THAT LUCKILY WE ARE
GOOD ON TIME IS THAT RIGHT NOW
ALL OF THE SLIDES I TELL YOU IS
IN HEX-STYLE, BECAUSE WHEN WE
THINK ABOUT THIS SCREEN, WE CAN
PERFORM THIS EXPERIMENT IN 12
PERFORMANCES OR WE CAN LIKE THE
CELL CULTURE AND THEN THE THERE
WILD-TYPE, SO CAN YOU ACTUALLY
SCALE IT IN TERMS OF CONDITIONS
QUITE WELL.
AND THAT'S IN HEK THAT'S
FEASIBLE BUT OF COURSE, WHEN YOU
THINK ABOUT THESE RECEPTORS THEY
ARE EXPRESSED IN MANY DIFFERENT
CELL TYPES AND THIS
PASTEURIZATION CAN ALSO COME
FROM THE SIGNALING IN THE
EXPRESSED CELLS AND THAT'S WE
ARE WORKING TOWARDS RIGHT TO USE
MORE SPECIALIZED CELLS AND WHAT
I WANT TO JUST SHOW, THIS KIND
OF IMAGE AND I HAVE THE PICTURE
OF MIKE HERE IN THE AUDIENCE, SO
WE ARE RIGHT NOW WHAT WE ARE
SETTING UP, WE HAVE SET IT UP IN
THE LAB, OUR IPSC DIRECT NEURONS
WHICH WE ARE EXCITED TO USE FOR
THIS STUDY, FOR THE NEURONAL
RECEPTORS AND WE USE A SYSTEM
THAT WAS REALLY NICELY
IMPLEMENTED BY MICHAEL WHERE WE
HAVE THIS INDUCIBLE
TRANSCRIPTION FACTOR NG N2, AND
CAN LEAD TO YOU KNOW CORTICALE
NEURONS WITHIN 21 DAYS AND PLUS,
AND THAT'S JUST AN IMAGE OF
THESE CELLS AND I HOPE IN A FEW
MONTHS I CAN SHOW SOME OF THE
PROTEOMIC DATA WITHIN ANY CELL,
ANY CELL SYSTEM AND IT'S A LOT
OF WORK BY ANGELINNA IN THE LAB
AND COULD ONLY BE POSSIBLE
BECAUSE THIS IS STEEP LEARNING
CURVE BECAUSE IN THE LAB WE HAVE
NEVER DONE IT BUT MICHAEL HAS
BEEN SHARING A LOT OF -- A LOT
OF HIS KNOWLEDGE TO GET THIS UP
AND RUNNING IN THE LAB, SO THANK
YOU MICHAEL FOR THIS, AND I AM
REALLY EXCITED WE CAN USE THIS
CELL, PROBABLY NOT AS BIG, BUT
WE HAVE TO SCALE UP A BIT BUT
I'M VERY POSITIVE THAT WE CAN --
WE CAN MOVE TOWARDS THIS CELL
SYSTEM, RIGHT IN AND IF YOU
THINK ABOUT IT, ONCE WE HAVE THE
DERIVED NEURONS BUT IF YOU'RE
ABLE TO DO THAT IN IPSC DERIVED
CELLS THEN YOU CAN INTEGRATE OUR
RECEPTOR OF INTEREST IN IPSCs
AND DIFFERENTIATE THEM INTO
DIFFERENT CELLS, AND LOOK ACROSS
DIFFERENT CELL TYPES SIGNALING.
SO THIS IS THE -- THIS IS THE
IDEA FOR THE FUTURE.
SO AGAIN, THIS IS THE VERSION
THERE I SHOWED YOU BEFORE THAT
WE ARE TRYING TO FOLLOW IN THE
LAB.
WE WANT TO STUDY WHAT DRIVES
SPECIFICITY OF GPCR SIGNALING
ACROSS DIFFERENT RECEPTORS,
ACROSS DIFFERENT LIGANDS BY
SIMPLY GENERATING DATA IN AN
EDUCATED WAY, RIGHT?
AND CORRELATING THIS WITH WITHIN
THE FUTURE WITH INVITRO
PHENOTYPES AND WHEN WE INTEGRATE
THE DATA WE HOPE IT CAN -- USING
COMPLIMENTARY APPROACHES AND CAN
REALLY TALKING ABOUT MECHANISM
AND MONITOR.
SO MY LAST SLIDE IS MOST
IMPORTANT.
I COULDN'T DO THIS WORK BY
MYSELF, I AM LUCKY THAT THE LAB
HAS ONLY BEEN ACTIVE 1 AND HALF
YEARS AND I HAVE A REALLY
FANTASTIC TEAM OF PEOPLE THAT
WORK WITH ME AND WE LIKE PROTEIN
NETWORKS THAT WE WORK A LOT OF
NETWORK BECAUSE WE DO A LOT OF
APPROACHES AND NEED DIFFERENT
KNOWLEDGE AND YEAH, I'M VERY
PROUD OF MY LAB, HOW WELL THEY
WORK TOGETHER AND ALSO WITH A
PREVIOUS LAB, WE WORKED TOGETHER
A LONG TIME AND A NUMBER OF THE
STUDIES AT THE BEGINNING THAT I
SHOWED YOU WERE GENERATED, THE
DATA WAS GENERATED BY A
FANTASTIC TEAM THAT I WAS
LEADING IN THE LAB AND WE HAVE
COLLABORATIONS WITH THE UCSF,
AND THE LAB THAT WE LOVE AND THE
BIGGER SCREEN THAT WE'RE NOW
DOING TOGETHER IN COLLABORATION
WITH THE BIO-X.
SO WITH THAT THANK YOU VERY MUCH
AND I AM HAPPY TO ANSWER ANY
QUESTIONS YOU MAY HAVE.
>> YES, ALEXANDRA?
>> I HAVE A QUESTION FOR THE
[INAUDIBLE QUESTION FROM
AUDIENCE -- AWAY FROM MIC ]
>> THAT'S RIGHT.
SO THAT'S A VERY GOOD POINT,
RIGHT?
WE HYPOTHESIZE THIS FROM PRIOR
KNOWLEDGE AND ALSO WE ARE NOW
PURIFYING THE RECEPTOR AND MATCH
THE PHOSPHORYLATION SITES AND
BACK THIS UP.
YEAH.
>> YEAH, ABSOLUTELY.
YEAH.
>> NO, NO, THIS IS A GOOD POINT
AND WE HAVE TRIED SOME INITIAL
EXPERIMENTS THAT HAVE NOT BEEN
SO -- JUST IN THE SENSE OF
GETTING THE RECEPTOR AND THEN WE
USE PROTEOMICS AND GPC Rs NOOD
OTHER ENZYMES SO THAT'S WHAT
WE'RE PLAYING AROUND WITH TO MAP
THESE SPECIFIC SIDES.
[LAUGHTER]
YEAH, THIS IS A VERY GOOD POINT.
IT'S ALL INDIRECT, RIGHT BUT WE
HAVE THEM AT THE PHOSPHORYLATION
SITE.
COMPELLING.
YEAH.
VERY GOOD POINT.
YEAH.
>> I HAVE A QUESTION --
[SPEAKING AT SAME TIME ]
[SPEAKING FOREIGN LANGUAGE ]
>> TWENTY-ONE, YES.
>> [INAUDIBLE QUESTION FROM
AUDIENCE -- AWAY FROM MIC ]
NYEAH, --
>> YEAH, THIS IS A BIAS COMPOUND
THAT IS BIAS FOR G-PROTEIN
SIGNALING OR IT INDUCES
G-PROTEIN SIGNALING OR AT LEAST
PARTIAL FOR SIGNALING BUT IT
DOESN'T RECRUIT
>> [INAUDIBLE QUESTION FROM
DIRECTLY
INTERNALIZE AND --
>> [INAUDIBLE QUESTION FROM
AUDIENCE -- AWAY FROM MIC ]
>> T130 IS A
BIASED LIGAND, IT'S NOT THIS 1
BUT THEY HAVE SHOWN ANALGESIC
EFFECT ACTIVATING G-PROTEIN
SIGNALING BUT THE IDEA WAS TO
DESIGN THIS BIAS LIGAND THAT
DON'T RECRUIT ARRESTIN AND DON'T
INTERNALIZE WITH THE HOPE THAT
THE ARRESTIN SIGNALING
ELIMINATING THEN, IF YOU BLOCK
THIS IT LIMITS ALL THE SIDE
EFFECTS, THAT'S NOT QUITE TRUE,
RIGHT?
BUT IT'S STILL AN ANALGESIC.
>> [INAUDIBLE QUESTION FROM
YEAH, MICHAEL?
>> [INAUDIBLE QUESTION FROM
AUDIENCE -- AWAY FROM MIC ]
BASED
>> GREAT QUESTION.
PART IS OPIOID RECEPTOR AND NOT
SURPRISINGLY REVIEW, LIKE YOU DO
IN TEXT TILES HOW DOES IT LOOK
IN NEURONS AND THE NEURAL TYPE.
AND THAT'S WHAT WE'RE STARTING
TO GET MORE SYSTEMATICALLY BUT
FOR THIS RECEPTOR WE USE BECAUSE
WE COULDN'T GROW THE NEURONS AT
THE TIME WE USE CELLS SUCH AS
NEUROBLASTOMA CELL LINE AND WE
ARE ALSO INTERESTED TO SEE HOW
LIKE, DOES THE RECEPTOR ENGAGE
SIMILAR PROTEOME, RIGHT?
AND IT WAS INTERESTING TO SEE
THAT THE MAJORITY OF INTERACTION
ARE CONSERVED BUT THERE ARE
DIFFERENCES.
FOR EXAMPLE, WE FOUND COM3, AND
IT'S VALIDATED IN THE LINE, AND
THE APPROXIMATE INTERACTION
NETWORK WE FOUND A DIFFERENT
NEURON, SO SAME FOR SOME OTHERS,
RIGHT?
THEY ARE RELATED BUT MAYBE THE
FRONT END AND THAT WILL BE
INTERESTING, WE HAVEN'T -- I
ACTUALLY HADN'T THOUGHT ABOUT
THAT BUT I THINK THIS WILL BE
INTERESTING, RIGHT, WHEN WE NOW
DO THE SCREEN ACROSS THESE
DIFFERENT GPC Rs TO LOOK, TO
LOOK BASED ON EXPRESSION DATA
RIGHT, ON DIFFERENT CELL TYPES
CAN IT CAN CONSERVED ON IT.
>> YEAH.
>> YEAH, I THINK THAT WOULD BE
INTERESTING AND I THINK
SPECIFICALLY WHEN WE -- IF WE
INITIALLY TART TO HAVE THESE
KIND OF DATA SETS AND SEE HOW
DIFFERENT THEY LOOK AND
DIFFERENT CELLS THEN WE CAN TEST
THIS IS IF YOU FIND THE CELLS
ENHANCED DIFFERENT EXPRESSION OF
THESE NETWORK COMPONENTS, DOES
IT LOOK DIFFERENT.
GREAT IDEA.
I WILL HOPEFULLY IN THE FUTURE
WE WILL FOLLOW UP ON THIS IDEA.
I LIKE IT.
YES, PLEASE.
>> REALLY WONDERFUL TALK, I HAVE
A QUESTION ABOUT THE APPROXIMATE
LABELING.
IF MY MEMORY'S CORRECT, APEX IS,
SIZE OF THE APEX IS QUITE BIG SO
IS THERE ANY EFFECT AT THE
LABELING OF THE APEX, YEAH, FOR
I BIOLOGICAL CHARACTERISTIC
CHANGES?
>> THE SO THE APEX I THINK IS
35, I'M NOT SURE THE NUMBER
CORRECTLY IN MY MIND, THAT CAN
AFFECT THE FUNCTION IF YOU
ATTACK A RECEPTOR WITH A LARGER
PROTEIN.
SO THERE ARE FEW WAYS HOW WE GO
ABOUT THIS.
THE INITIAL STEP IS WE USE A
FLEXIBLE LINKER WITH THE HOPE
RIGHT THAT THIS APEX CAN MOVE
AROUND AND MAKE SPACE FOR
INTERACTING PROTEIN, AND THEN
OBVIOUSLY WE TASK FUNCTIONALITY
OF THE RECEPTOR AFTER -- FOR
EXAMPLE, A GOOD APPROXIMATION IS
WHEN WE COMPARE THE APEX
RECEPTOR AND A NONATTACK
RECEPTOR WE CAN LOOK AT THE
SIGNALING OUTCOME THEY PRODUCE,
LOOK AT TRAJECTORY, CAN SEE IS
IT SIMILAR TO THE WILD-TYPE
RECEPTOR OR DOES IT ACTUAL LYE
EXPRESSOT CELL SURFACE, RIGHT?
AND THIS IS FOR APPROXIMATION TO
SAY, OKAY, THE FUNCTION OF THE
RECEPTOR MIGHT NOT BE IMPAIRED,
RIGHT?
THERE'S NO GUARANTEE, BUT THESE
ARE THE ASSPECS WE TEST FOR FOR
FUNCTIONALITY.
AND WHAT WE SEE IS THE RECEPTOR,
SO THERE ARE SOME GFCRs THAT
HAVE THIS PDC BINDINGA THE TAIL
OF THE RECEPTORS AND THESE CAN
BIND PDC BINDING PROTEINS THAT
MEDIATE TRAFFICKING AND WHAT WE
HAVE SEEN FOR, THE BETA 2
RECEPTOR IS A CLASSIC EXAMPLE
WHEN WE USE IF, APEX DIRECTLY TO
THE C-TERMINAL TAIL, WE SEE THAT
THERE IS A DIFFERENCE COMPARED
TO THE NONTAGGED RECEPTOR IN
TERMS OF TRAFFICKING AND THIS
WAS AN INITIAL PUBLICATION WHEN
WE TESTED THE APEX, AND WE
ALWAYS START WITH THE BETA
RECEPTOR BECAUSE IT'S A WELL
CHARACTERIZED GPCR RECEPTOR, AND
WE PLACE IT INTO THE TAIL, WE
CHOP THE TAIL APART AND PUT IT
BETWEEN FUNCTIONAL GROUPS AND
LEAVE THE PDC DOMAIN FREE, THEN
IT SHOWS SIMILAR FUNCTION IN THE
WILD-TYPE RECEPTOR, SO MEANING
IT'S A CONCERN, HOWEVER, I WANT
TO SAY WITH THE LONG ANSWER TO
SAYING YES, THERE IS A CONCERN
THAT CAN CAN EFFECT
FUNCTIONALITY AND IT'S IMPORTANT
TO TEST THIS AND WE'RE TRYING TO
APPROXIMATE THIS SCIENCE BY
USING FUNCTIONAL ASSAYS.
>> THANK YOU.
>> YES.
>> -- MIGHT HAVE BEEN G-PROTEIN
AND I WONDER WHETHER YOU OBSERVE
SIMILAR PATTERNS WITH YOUR
RECEPTOR AS OPPOSE TO DOMINANT
PROTEIN AMONG THOSE RELATED
RECEPTORS?
>> MM-HMM.
THAT IS CORRECT.
SO WHAT I HAVE TO SAY IS THAT,
YOU KNOW OUR METHOD IS REALLY
GOOD TO SEE PROTEINS THAT ARE
NOT CLOSE IN PROXIMITY OF THE
RECEPTOR BEFORE ACTIVATION AND
WHEN THE RECEPTOR IS ACTIVATED
CAN BE RECRUITED RIGHT?
BECAUSE THEY'RE CHANGING OF --
LIKE PROXIMITY AND THAT'S WHAT
WE LABELED SO THE G-PROTEINS
LIKE WE LABEL ALL THE G-PROTEINS
BUT THEY'RE ALREADY IN CLOSE
PROXIMITY OF THE RECEPTOR BEFORE
ACTIVATION, SO WE DON'T SEE,
LIKE FOR EXAMPLE, WHEN WE
ACTIVATE THE NEW OPIOID SYSTEM,
WE DON'T SEE A DIFFERENCE OF
LABELING IN THE INHIBITOR
G-PROTEIN BECAUSE WE JUST DON'T
HAVE -- WE DON'T HAVE THE
TEMPORAL RESOLUTION BUT ALSO NOT
THE SPATIAL RESOLUTION, THEY'RE
ALREADY CLOSE IN THE
NONACTIVATED SO WHAT WE CAN ONLY
DEDUCE IS FROM THE DOWN STREAM
EFFECTS THAT WE SEE, SO FOR
EXAMPLE, WE FOUND WITH THE NEW
RECEPTOR, AND SPECIFICALLY, IT'S
ASSOCIATED WITH -- IT'S ACTUALLY
NOT INTERACTING WITH THE
RECEPTOR, IT'S INTERACTING WITH
THE REDUCED GENOME, AND WE SEE
FOR MANY GI RECEPTOR WE SEE ALL
COMING UP SO THIS GOOD.
SO INDIRECT EFFECTS WE CAN READ
OUT FOR THE G-PROTEIN SIGNALING
BUT THE DIRECT PROTEIN WE DON'T
SEE.
NOW YOU ASK IF WE CAN SEE
DIFFERENCES AND IT'S TRUE THAT
WHEN 1 OF THE RECEPTORS THAT WE
TESTED ACROSS THE RECEPTORS IS
KNOWN TO PATHWAY GQ AND GI AND
YOU COULD SEE THAT WE SEE
NETWORK COMPONENTS THAT ARE
RELATED TO CONSTANT ACTIVATION
AND WE SEE PREDICTORS LIKE EY4
FOR EXAMPLE, SO THIS SAY MERGE
BETWEEN GI AND GQ IN TERMS OF
THE INTERACTION NETWORK.
SO INDIRECTLY WE CAN MAYBE
OBSERVE IT BUT YOU KNOW THAT'S A
SHORT COMING OF THE THE METHOD,
IT'S NOT THE MAGIC BULLET,
THINGS THAT ARE CLOAT BY THE
RECEPTOR ACTIVATION, WE CAN
LABEL BUT WE CANNOT SEE THE
DIFFERENCE, BASICALLY WITH MOST
OF THE RECEPTORS WE CAN LABEL
ALL GENE PROTEINS BASICALLY.
>> WOULD YOU CONCLIEWT FROM THIS
THAT THERE'S PREASSEM MYELIN
ALREADY OF RECEPTOR PROTEIN OR
JUST SUCH A HIGH DENSITY OF
G-PROTEIN IN THE CELL?
>> I THINK THAT'S HARD TO
CONCLUDE WHAT IT IS.
I WOULD SAY THEY'RE JUST CLOSE
BY AND IF THEY ARE PREEXAMINED
OR NOT, IT'S JUST NOT -- YOU
KNOW IT DOESN'T HAVE THE
RESOLUTION TO TELL.
SO IT'S HARD TO -- I MEAN I WISH
WE COULD SAY THAT BUT IT'S NOT
THE MAGIC BULLET THAT ANSWERS
ALL THE QUESTIONS, YEAH?
AND THERE'S LIMITATION, RIGHT?
THANK YOU SO MUCH.
IF THERE ARE NO FURTHER
QUESTIONS, THANKS FOR THE GOOD
DISCUSSION AND THANK YOU FOR
COMING.