G Protein Coupled Receptors
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Lindau Nobel Laureate Meetings293 / 340
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NobeliumChemistryRezeptorProteinAzo couplingTopicityG proteinLecture/Conference
00:35
PsychopharmakonRezeptorProteinHistamineOpioidPolymorphism (biology)Medical prescription11 (number)Block (periodic table)ThylakoidNobeliumOrphan DrugAgonistSerotoninG proteinAzo couplingRezeptorProteinCell membraneGeneSeparation processProcess (computing)PhysiologyGrease interceptorGesundheitsstörungLecture/Conference
01:03
RezeptorAgonistOrphan DrugPsychopharmakonMedical prescriptionSerotoninHistamineOpioidWine tasting descriptorsNobeliumHomocysteinePhysiologyAmpicillinStimulus (physiology)Activation energyRhodopsinRetinalLigandConformational isomerismBioavailabilityProteinLigandHighway Addressable Remote Transducer ProtocolBinding energyRezeptorReaction mechanismSingulettzustandProteinAgonistAdenylate cyclaseConformational isomerismAdrenergic receptorPharmacologyActivity (UML)ErdrutschG proteinPharmaceutical drugPhysiologyExtracellularLigandPeptide sequenceBiosynthesisOpioidRecreational drug usePhlegmatizedEnzymeMoleculePhosphorylationProcess (computing)Anomalie <Medizin>C-terminusThermoformingSecond messenger systemActive siteSystemic therapyChemical elementNobeliumWursthülleProtein domainGeneSetzen <Verfahrenstechnik>ConcentrateProtein subunitHomologisierungBiological membranePeriodateBase (chemistry)Functional groupErdölraffinationAngiotensinTandem-ReaktionVasoconstrictionKlinische ChemieBiomolecular structureCell (biology)LigandPeptideMachinabilityIce frontGrease interceptorChemical propertyArzneimitteldosisSeparation processCardiac arrestOrlistatAzo couplingBeta sheetTransmissivität <Hydrologie>Stimulus (physiology)OppressionEpinephrineMeat analogueIntergranular corrosionAngiotensin-converting enzymeProlineEpidermal growth factorPhosphorous acidGrade retentionTrauma (medicine)AgeingLeadIndiumLecture/Conference
11:00
MedroxyprogesteroneAgonistMorphineWine tasting descriptorsRezeptorAngiotensinPressurePharmacyWaterfallHondurasGolgi apparatusTiermodellAction potentialStorm surgeLigandPharmacologyAllosteric regulationActive siteBinding energyPsychopharmakonG protein-coupled receptorConformational isomerismFood additiveAngiotensinAmino acidBinding energyAgonistPharmaceutical drugChemical propertyActivity (UML)RezeptorWursthülleLigandAngiotensin II receptor antagonistAllosteric regulationProcess (computing)Recreational drug useSystemic therapyMeat analoguePeptideKlinische ChemieChemical compoundMoleculeVasoconstrictionOpiatrezeptorAreaGolgi apparatusConcentrateConformational isomerismG proteinOpioidAnalgesicAzo couplingDeterrence (legal)TiermodellLactitolLigandSerotoninBase (chemistry)Conformational changeActive siteTool steelMacromoleculeSetzen <Verfahrenstechnik>Reaction mechanismKlinisches ExperimentMetabolic pathwayCooperativityWine tasting descriptorsThermoformingBlock (periodic table)PainProteinSubstitutionsreaktionResidue (chemistry)LeadPan (magazine)BranntweinCell growthModul <Membranverfahren>Trauma (medicine)StimulantOperonHistamine antagonistPressureTachyphylaxieSingulettzustandAtom probeGrease interceptorBeta sheetCombine harvesterLecture/Conference
20:47
RezeptorMoleculeAllosteric regulationLaundry detergentMedroxyprogesteroneLibrary (computing)Binding energyDimethylsulfoxidDarmstadtiumAgonistSurface scienceChemical compoundActivity (UML)ProteinCyclopentanonArzneimitteldosisLibrary (computing)LigandAction potentialStimulantRezeptorSingulettzustandBeta-2 adrenergic receptorChemical compoundIsoprenalinActive siteErdölraffinationConcentrateGene expressionWursthülleAsthmaAgonistAntibodies (film)Binding energyCell (biology)GesundheitsstörungMoleculeBiological membraneAllosteric regulationFunctional groupBeta sheetG proteinConformational isomerismThermoformingCooperativityIsotopenmarkierungAdrenergic receptorHelicität <Chemie>Surface scienceHelixCytoplasmaCell membraneSetzen <Verfahrenstechnik>Alpha particleReaction mechanismStereoselectivityCardiac arrestProtein domainAzo couplingLeft-wing politicsMultiprotein complexSignal transductionSense DistrictDesorptionWine tasting descriptorsKlinisches ExperimentTiermodellGrease interceptorDissoziationskonstanteWater purificationCocaIceAnaerobic digestionTool steelElectronegativityModul <Membranverfahren>Activity (UML)AgeingMalerfarbeGas cylinderProteinEpinephrineSynthetic oilArzneimitteldosisEnzyme inhibitorRecreational drug useTranslation <Genetik>Storage tankBase (chemistry)Chain (unit)WaterRepeated sequence (DNA)Click chemistry
30:35
Binding energyActivity (UML)Allosteric regulationRezeptorSet (abstract data type)Klinisches ExperimentTranslation <Genetik>Base (chemistry)Wine tasting descriptorsLibrary (computing)Surface finishingWursthülleLecture/Conference
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NobeliumLecture/ConferenceComputer animation
Transcript: English(auto-generated)
00:14
Good afternoon, pleasure to be with you. So my topic today, G-protein coupled receptors,
00:24
has been one I've been fascinated by and deeply involved in trying to study for my entire research career, which actually began, coincidentally, 50 years ago, almost to the day when I arrived at the National Institutes of Health as a young physician,
00:42
trying to find out whether I might also enjoy doing research. That was July 1, 1968. So the G-protein coupled receptors, which are also known as seven transmembrane spanning receptors, represent by far the largest, the most versatile, and the most ubiquitous of the several families
01:01
of plasma membrane receptors. There are anywhere from 800 to 1,000 genes in this superfamily, and they regulate virtually all known physiological processes in humans. And this includes, by the way, several of our sensory modalities, such as vision, taste, and smell.
01:21
But most importantly, from the point of view of clinical medicine, the G-protein coupled receptors represent by far the largest single class of therapeutic drug targets. A few examples are listed on the slide, include adrenergic receptor agonists and antagonists,
01:41
antihistamines, and opioids as just several examples. Now this slide, which is taken from Earl Sutherland's Nobel lecture in 1971, reasonably depicts the state of the field when I began my own work 50 years ago. As you can see, Sutherland identifies
02:03
the adenyl cyclase enzyme, which he had discovered, and which catalyzes the synthesis of the second messenger cyclic AMP. He conceptualizes it as the central target of extracellular drugs, hormones, and neurotransmitters.
02:21
Notice that there is absolutely no evidence in his conception of an independent receptor entity. In fact, at the time, there was a great deal of controversy and skepticism as to whether such receptor molecules might exist. So let's fast forward 20 years to the early 90s,
02:44
after 20 years of work, and see where those efforts had brought us in our quest to understand this black box of hormone and drug signaling. During this period of time, we had managed to establish this large family of seven membrane spanning
03:05
G-protein coupled receptors. We did this by a long and laborious process in which we first developed radioligand binding techniques which allowed us to demonstrate the existence of these binding sites. We solubilized them, we purified them,
03:22
we obtained small pieces of amino acid sequence from micro-sequencing and used this to clone the genes and see DNAs, first for the beta-2 adrenergic receptor, and then for other adrenergic receptors, meaning receptors which bind adrenaline, and ultimately for more than a dozen
03:41
other seven membrane spanning receptors. We noticed their strong homology and architectural analogy, both with each other and with the previously sequenced visual protein rhodopsin, thus realizing and establishing that there was this large family of receptors. Contemporaneously, Rodbell and Gilman
04:03
had discovered that the major mechanism, or I should say a major mechanism of action of the receptors, the paradigmatic mechanism from which they derived their names was to activate heterotrimeric G-proteins, leading to cascades of protein phosphorylation.
04:21
For this work, they received the Nobel Prize in Physiology or Medicine in 1994. Now contemporaneously with all of this work, I had been fascinated with a problem throughout my research career, and it referred to a fairly universal phenomenon known as desensitization,
04:42
illustrated in the lower part of the slide. This refers to the almost universal phenomenon that when you stimulate one of these receptors with an agonist, such as the beta-adrenergic receptor with adrenaline, the response, in this case, cyclic AMP generation, rapidly wanes, even in the continuing presence of the stimulus.
05:05
A 20 or 25-year effort to understand what the mechanism of this was led us to the discovery of two small families of proteins. The first we call G-protein coupled receptor kinases, or GRKs. They phosphorylate the receptors on multiple sites
05:22
on their carboxy terminus. Importantly, they only phosphorylate the activated form of the receptor. The second family of proteins are called beta-arrestins, or arrestins. They bind to the phosphorylated receptor and generally only to the phosphorylated receptor
05:42
and thereby somehow lead to desensitization of the receptors. We speculated at the time and subsequently have proven with structural studies, which I don't have time to review today, that this led to desensitization of the receptors by sterically interdicting the binding
06:02
of the G-protein to the receptor, essentially in a competitive fashion. Today we know that there are seven members of the GRK family, of which there are crystal structures now for most, but only four of these, GRKs 2, 3, 5, and 6,
06:22
are universally distributed. GRKs 1 and 7 are found only in the retina. This crystal structure from the laboratory of John Tesmer in collaboration with my lab 15 years ago, shows the conserved fold of the kinases with a central catalytic kinase domain flanked by two regulatory domains.
06:42
And in the case of GRK2, down to the beta-gamma subunits of the G-proteins. There are only four arrestin genes, and only two of them, beta-arrestin 1 and beta-arrestin 2, are universally distributed. Alas, these are somewhat confusingly
07:00
also known as arrestin 2 and arrestin 3, respectively. Now, as I mentioned, we discovered the arrestins because of their role in desensitization. But about five years later, it was discovered that the beta-arrestins also were able to serve as adapters,
07:20
which connect the receptors to clathrin, AP2, and other elements of the endocybic machine, leading to internalization of the receptors, which is a virtually universal process. Then, about 10 years after their discovery, we found, very surprisingly,
07:40
that the beta-arrestins, which we had discovered by virtue of their ability to turn off or desensitize G-protein signaling, were actually a signaling system in their own right, and that even as they turned off G-protein signaling, they could serve as adapters to connect the receptors directly to a variety of enzyme signaling systems,
08:02
leading to a variety of cellular physiological outcomes. Thus, the arrestins were able to serve three distinct functions, desensitization of G-proteins, internalization, and activation of signaling in their own right.
08:21
Then we made an even more surprising discovery, and that was that some ligands, meaning drugs or hormones which can bind to the receptors, were actually able to stimulate signaling, not by both of these processes, but only by one or the other.
08:42
That is to say, either G-protein or beta-arrestin. We called such ligands, which could only signal in one of those two ways, biased ligands. So to go over that definition, a biased agonist is a ligand which stabilizes a particular act of confirmation
09:00
of a receptor, thus stimulating some responses, but not others. Now, this was a very surprising discovery for us and for others in the field. For several reasons. In terms of basic fundamental receptor theory, the general idea prior to that time
09:20
had been that receptors can exist in two conformations, inactive, generally referred to as R, and active, R star, which were in equilibrium, an equilibrium which was far to the left in the absence of a stimulant. But an agonist, A, was viewed as having much higher affinity
09:42
for the act of R star form, to which it preferentially bound, thus pulling the equilibrium to the right, to the active state. And then all downstream signaling was viewed as some function of the concentration of A R star. But the idea that a ligand could bind to the receptor
10:03
and stimulate one type of signaling and not the other is formally inconsistent with that idea, and virtually demands that there must be multiple active conformations at minimum, and, surely, we now know, marked over simplification,
10:21
two conformations, one coupled to G protein, one coupled to beta-resti. Let me show you, in a simple cellular system, what biased signaling would look like. So this is a simple cultured cell system which has been transfected with a typical GPCR,
10:40
that for the octapeptide vasopressor angiotensin. In this experiment, we're stimulating the receptors with angiotensin itself, Ang-2, the endogenous ligand for this receptor. We're measuring two things, in red, an output due to G protein signaling, and in black, an output due to beta-restin
11:03
recruitment and signaling. Notice that both of these dose-response curves, in response to increasing concentrations of angiotensin, are very similar. Angiotensin is what we call a biased, I'm sorry, a non-biased or balanced ligand, which has equal ability to stimulate both pathways.
11:25
ARB stands for angiotensin receptor blocker. These are very important drugs in clinical medicine, used all the time. There may be people among the laureates who are taking an ARB, and they, of course, don't signal at all.
11:40
They're blockers, they're conventional blockers, similar to beta blockers or antihistamines, and you can see that they stimulate in either process. But consider this compound, 12-0027, an experimental compound. It is an octapeptide analog of angiotensin with three of the eight amino acid residues substituted.
12:04
Notice in red that it has absolutely no ability to activate the system for G protein, in contrast with angiotensin. But it is able to activate barrestin pathway, fairly robustly. So this is an example of a barrestin-biased ligand.
12:24
It cannot activate G protein, and in fact, with respect to G protein, it is an antagonist. If you tested it for G protein signaling, you would conclude that it is only an antagonist. But if you had assays to test for beta-arrestin signaling,
12:41
which we've only had for about 12 years now, you would realize that it has agonist properties. The discovery of biased agonism has very important therapeutic consequences as well, because if we can make biased drugs, they may have much greater specificity and effectiveness.
13:03
Let me give you a couple of examples of how this would be true. Consider the mu opioid receptor, a typical GPCR, which is the target of all the opiates that we use therapeutically, and which are, alas, being abused very greatly throughout the world.
13:22
All the pain-relieving effects of opiates and related compounds are mediated by the mu opioid receptor. We know that these therapeutic effects, the analgesic effects, are mediated by signaling through the G protein signal, G sub I, to be specific.
13:45
But there are other unwanted side effects, some of them annoying, some lethal, of signaling through the mu opioid receptor, such as constipation, annoying, respiratory depression,
14:02
lethal, and tolerance, that is the need for increasing concentrations of the drug the more you take them, all of which we were able to show some years ago are mediated through beta-arrestin signaling, and which are lost in mice which have been deleted for barrestin-2.
14:22
So one might speculate that if one had a G-biased ligand for the mu opioid receptor which was able to signal through G proteins and hence give us the anti-nociceptive or pain-relieving effects, but which could not signal through barrestin
14:42
that one would have a drug which had the ability to have therapeutic effects with much fewer side effects, including less of the lethal respiratory depression. In fact, this turns out to be true, both in animal models and in humans,
15:00
and the first such compound is currently before the FDA for approval. Now, depending on which arm of the signaling system mediates what we would view in a therapeutic context as a therapeutic effect, and which arm mediates what we would view
15:22
as adverse ill effects, will determine what direction of bias one might want for a particular drug. For example, in the case of the angiotensin receptor, its G protein mediated effects lead to vasoconstriction and hypertension, and it is those effects
15:41
that we would like to block therapeutically in patients with hypertension and heart failure. In fact, ARBs which block this are amongst the most commonly prescribed drugs in cardiovascular medicine. However, ARBs also block the barrestin arm of signaling, and there's some evidence that that arm of signaling
16:03
is potentially useful. For example, it has inotropic effects and anti-apoptotic effects. So here one might speculate that a drug which was beta-restin biased, which blocked G protein signaling, but which facilitated or permitted barrestin signaling
16:21
might be useful. For example, in the case of hypertension or heart failure. Again, this turns out to be true, and in animal models, such a compound, O2-7, has been shown to slow the progression of heart failure in animal models to lower blood pressure, increase cardiac performance,
16:40
and to have anti-apoptotic effects. Again, such compounds are currently in clinical trials. Now, biased ligands is one example of some potentially important clinical therapeutic progress and developments which have come
17:02
from some of this basic research. But I wanna give you another example of an area which I think in the coming years will be of great importance, and that has to do with so-called allosteric drugs. So first, a definition or two. We refer to the orthosteric binding site on a receptor
17:22
as being that site to which the endogenous agonist normally binds. It's where adrenaline binds on the adrenergic receptors, where serotonin binds on the serotonin receptor, et cetera. But there also, in theory, and it turns out in reality,
17:41
can be allosteric ligands. These are ligands which bind anywhere else on the receptor. An allosteric ligand binding to the receptor may or may not change its conformation, and depending on how it does so will depend on what effects it might have. For example, it might lead to signaling in its own right,
18:03
or it might enhance signaling by an orthosteric agonist. We would call this a positive allosteric modulator, or PAM. Or it might diminish signaling by an orthosteric agonist. We would call this a negative allosteric modulator.
18:26
What I want to tell you about now is how we're currently trying to develop such drugs, but for two reasons. First, we're interested in trying to understand the molecular details of the conformations of the receptor,
18:42
which are responsible for things such as biased signaling, but also which might lead to allosteric mechanisms of signaling. So one reason for trying to obtain novel allosteric molecules is to help us understand specific conformational changes
19:03
in the receptor. The other, of course, is to obtain novel types of drugs. Now, the interest in allosteric modulators, again, stems from these dual interests, in the basic research interest and in the clinical interest, which, in my own case, go hand in hand.
19:22
So why might they be useful as basic research probes? Well, it has to do with the fact that, as I mentioned, we now understand that the receptors can isomerize and basically occupy many different conformations. And that's true whether the receptors
19:41
are in their inactive state or even when they are in their active state and occupied by very strong high-affinity agonists. But as we heard from Dr. Frank this morning, multiple conformations is a real problem for studying structures of macromolecules.
20:03
It's always easier if you can isolate a particular conformation, although with cryo-EM, as we heard, it's now becoming more possible to study multiple conformations simultaneously. So one interest in obtaining these allosteric molecules
20:20
as tool compounds is that often, if one combines an orthosteric molecule in the orthosteric site with an allosteric molecule binding at some remote site, often the cooperativity between those two binding events will lock the receptor into a particular conformation.
20:44
And that particular conformation is more accessible to us for study, on the one hand, and may also be specialized for a particular biological and clinical effect, which is also something we might want. So let me show you how we are attempting
21:02
to obtain and develop a whole toolkit of novel allosteric molecules. In order to do this, we have adapted affinity-based selection methods to the GPCRs for the first time. We're taking three approaches. First, we're using nanobody phage-display libraries.
21:23
Remember that nanobodies are single-variable antibody domains which we obtain from heavy-chain-only antibodies obtained from llamas immunized with receptor preparations. We can also use RNA-aptoma libraries containing as many as 10 to the 15th different molecules.
21:42
And we can also use huge DNA-encoded small-molecule libraries containing up to one billion small molecules. Let me repeat that. One B with a billion, one billion molecules. In the interest of time, I want to talk only about these DNA-encoded small-molecule libraries.
22:03
So for all three of these approaches, the basic approach is the same. We start with a purified receptor, and the examples I'm going to give you now have to do with the beta-2 adrenergic receptor as a model. We purify the receptor to homogeneity after overexpression.
22:20
We immobilize it on beads. We incubate that receptor with one of these very large libraries. We wash away molecules which don't bind. We take the molecules which have bound to the receptor, we elute them, we go back over and over and over again
22:43
with multiple rounds of adsorption and desorption until the binders are sufficiently enriched that, in the case of our DNA-encoded small-molecule libraries, such that they can, by deep sequencing, be identified and then characterized.
23:03
A nice thing about this approach is that we can bias, and I'm using bias in a different sense now, we can bias the selections by either selecting or counter-selecting against receptors which have, for example,
23:20
an orthosteric agonist or antagonist on the site, thus favoring either positive or negative allosteric modulators, or even by forming complexes of the receptor with one of its transducers, G protein or beta-arrestin, thus biasing us in the direction of obtaining biased ligands.
23:41
Let me give you a couple of examples. So, when we use the beta-2-adrenergic receptor as our target, either unoccupied by any ligand or with an antagonist on it, most of the receptors will be in an inactive conformation. One might think one would pull out negative allosteric modulators.
24:03
That turns out to be true. An example is this compound we recently isolated, which we called compound 15. So, here are some very classical cellular signaling experiments, in this case using a cultured cell line
24:22
expressing the beta-2-adrenergic receptor. Of course, the classic biochemical response to beta-adrenergic stimulation, which has been utilized in studies like this for many decades, is cyclic AMP generation. So, if we stimulate the cells with ISO, an abbreviation for isoproterenol, a synthetic adrenaline-like drug,
24:45
we get a nice dose-response curve of stimulation. But as we add increasing concentrations of compound 15, the curve shifts to the right to lower affinity and lower maximum. If we do a competition radioligand binding experiment shown on the right,
25:07
isoproterenol inhibits the binding of a radiolabeled antagonist. They're competing for the orthosteric site. But the addition of compound 15 is able to progressively shift that curve to the right. It lowers agonist affinity.
25:25
Here's a classical example of cooperativity. Two forms of radioligand binding to the beta-adrenergic receptor. In red, the binding of a radiolabeled antagonist. In blue, the binding of a radiolabeled agonist.
25:41
Notice that in the presence of increasing concentrations of compound 15, the binding of the radiolabeled antagonist increases, that of the agonist decreases. Compound 15 would be said to be negatively cooperative with the agonist, positively cooperative with the antagonist.
26:00
It is a classic negative allosteric modulator of the beta receptor. And in fact, the very first allosteric beta blocker, if you will. All other agonists or antagonists of the beta receptor. Or for that matter, virtually all GPCRs at this point are orthosteric ligands.
26:21
How does it work? We know it's not binding to the orthosteric site, but how does it work? So to understand that, we solved a structure, a 2.8 angstrom, of the beta-2-adrenergic receptor, shown on the left, bound simultaneously to an orthosteric antagonist,
26:41
a beta blocker called carazolol, and compound 15. So for those of you not used to looking at crystal structures, this is a classical ribbon diagram of the beta-2-adrenergic structure that was originally obtained by the Kobilka group a number of years ago.
27:02
You can see the seven alpha-helical membrane spanning domains. Notice, to our surprise, that this very first allosteric beta blocker is actually binding on the other side of the plasma membrane. It is in fact in a novel allosteric pocket not known to be there before.
27:22
These two sketches on the right, these two cartoons, demonstrate views of the structure of the receptor as if one were inside the cell. So pretend you're in the cytoplasm of the cell, looking up at the plasma membrane, you would see this inner surface of the receptor
27:40
and buried in this little pocket, which, by the way, is part of the site to which the G protein likes to fit, is our compound 15. Now look at this yet another representation, where each transmembrane span is a helix, is shown as a cylinder.
28:01
You can see compound 15 interdigitated deeply amongst the various helices at the inner surface of the receptor. Notice that it makes extensive contacts with the inner surface of transmembrane one, two, six, seven,
28:20
and even this non-transmembrane short alpha helix eight. Now, normally when the receptor is activated by an agonist binding here, the helices splay apart, allowing the G protein or arrestin to fit in, something like this. So you can see the movement, for example, which is greatest with helix six here.
28:48
But in the presence of compound six, 15 rather, this is very difficult, because it's like a piece of chewing gum, if you will. It's making contacts with all these membrane spans,
29:01
and it is holding them together, preventing them from being activated to allow the transducers to fit in. So it's a totally different mechanism. Competitive antagonism is the mechanism at the orthosteric site, but here we have an allosteric mechanism. Now let me just conclude by saying we think and we have evidence
29:21
that these procedures are quite universal. For example, if we use as our target the beta receptor occupied by a very strong agonist, now we obtain only positive allosteric modulators. For example, notice that for the same types of simple experiments I showed you before, we now get a completely opposite response.
29:44
So here's the isoproterenol cyclic AMP stimulation curve. Remember, compound 15 decreased this. Now compound six elevates everything. Here's an assay of beta arrest and recruitment. Isoproterenol leads to beta arrest and recruitment to the receptor.
30:01
Compound six progressively enhances the ability of isoproterenol to do that. So it is a positive allosteric modulator. Similarly, whereas compound 15 progressively diminished agonist binding affinity, compound six progressively increases it,
30:21
and shifts the competition curve to the left. So it's a classic positive allosteric modulator. Again, there's not time to discuss it, but again, a number of potential applications of that in asthma and other conditions. So to finish up, I just want to leave you with the idea, which I think will play out repeatedly in the laureate talks you're hearing this week,
30:43
that the distinction between basic or fundamental scientific research and translational and clinical research may be smaller than you think. Virtually every set of basic fundamental discoveries that one makes in the laboratory can potentially be leveraged and manipulated to clinical advantage.
31:05
And the work that we've been doing on receptors over these many decades has in fact borne very gratifying, and in many cases, very unexpected clinical fruit. And so I leave the young scientists with their thought, and I'll finish just by thanking the many people in my own laboratory,
31:24
shown here, and some of our collaborating laboratories, such as Brian Kobelka, with whom I shared the prize in 2012, and Jurgos Giniotis. Thank you very much.