Robert Lefkowitz (Duke University) Part 2: Beta-arrestins

I’m Bob Lefkowitz, an investigator of the Howard Hughes Medical Institute and a professor of medicine and biochemistry at Duke University Medical Center in Durham, North Carolina Today, I’m talking to you about drug and hormone receptors, and in particular, about one remarkable, huge family of such receptors, called the seven transmembrane receptors, also known as the G-protein coupled receptors In my first lecture, I told you in some detail about the history of how this field evolved and how we traveled from being quite skeptical about the mere existence of these receptors, to a fairly detailed understanding of how they function Today, I want to bring you right up to the minute, talking about interesting and exciting discoveries over the last five to ten years, which are completely reshaping our understanding of how the receptors function, and are regulated, and which can be leveraged to develop entirely new types of drugs The focus is going to be on a class of molecules called beta-arrestins Now, I introduced this in the first lecture, but to just briefly review, the classical signaling paradigm for understanding how G-protein coupled receptors work is as shown here, and was derived over the years largely from studies on two model systems: the beta-2 adrenergic receptor, for catecholamines, like adrenaline and noradrenaline, and the so-called visual receptor, rhodopsin, for which the stimulant is really photons of light Now, the key feature, as shown here, is that when the receptor is activated, by adrenaline or by a photon of light, it changes conformation, becomes activated, stimulates G-proteins, leading to activation of second messenger-generating enzymes, and those second messengers activate kinases leading to cellular responses But as we talked about, an opposing system immediately is engaged, which tends to shut down, or desensitize receptor action This involves a two-step mechanism. In the first step, a G-protein coupled receptor kinase, such as GRK2, shown here, phosphorylates the active conformation of the receptor, leading to a second step, in which a beta-arrestin molecule binds to the phosphorylated receptor, physically blocks G-protein coupling, sterically interdicts it, thereby leading to desensitization But the exciting thing in this field, which has developed over the last few years, is that an entirely new signaling paradigm is emerging And that is shown here. The notion is that the GRK/beta-arrestin system is really bi-functional At the very same time that it desensitizes G-protein signaling, it also serves as a signaling unit, a signal transducer, in its own right, which is able to lead to signaling to a growing list of biochemical pathways, some of which are listed here, which in turn lead to a variety of important cellular, physiological consequences This system also can lead to endocytosis of the receptors through clathrin So, today, we’ll be talking about this new appreciation of the signaling and other functions of beta-arrestins, and how this can be leveraged for drug discovery Let’s start with the first, or core function, of the beta-arrestins, which I’ve told you about already: their ability to sterically block G-protein signaling, thereby slowing the rate of second messenger generation But quite recently, we discovered, that this mechanism is actually more complex At the very same time that the arrestins are slowing the rate of G-protein activation, they also serve as adapters which can recruit the very enzymes responsible for the degradation of the second messenger Now, in the case of the beta receptor and Gs, which leads to cyclic AMP stimulation, that would be the cyclic AMP phosphodiesterase, shown here In the case of a Gq coupled receptor, like the muscarinic cholinergic receptor, which leads to diacyl glycerol formation, that would lead to the recruitment of diacyl glycerol kinase by beta arrestin Now, this function essentially serves to bring these degradative enzymes into close proximity with those microdomains of the plasma membrane where the second messenger is being generated

So, this new information suggests that even the core function of the beta-arrestins, the desensitization, is actually more complex than we originally perceived Specifically, it involves a concerted action whereby the beta arrestins both slow the rate of second messenger generation, while enhancing the rate of degradation Now, the second function of the beta-arrestins is to serve as adapters which facilitate the clathrin mediated endocytosis of the receptors in response to agonist This core function requires the ubiquinitation of beta-arrestins by E3 ubiquitin ligases, MDM-2 in particular is important here And that leads to the interaction of beta-arrestins, as an adapter, not only with clathrin, but with the clathrin adapter AP-2 and with a growing list of other elements of the endocytic machine Now, several years ago, we were carrying out a proteomics screen, which I’ll tell you more about in a few minutes, looking for molecules which bound to beta-arrestins. And in the course of doing that screen, Kunhong Xiao in our group discovered that a variety of cellular motors bound to the beta-arrestins, in particular, as one example, the kinesin, Kif3a This led to a very interesting line of research in our laboratory by Jeff Kovacs which involves Smoothened Now, Smoothened, as some of you may know, is a very atypical, non-canonical, if you will, seven-transmembrane receptor which may not even couple to G-proteins at all Together with its inhibitory co-receptor, Patched, it mediates the important effects of the ligand Sonic hedgehog, to lead to cellular proliferation and embryonic pattern formation Now, several years ago, a number of laboratories showed that in order for Smoothened to function, it must be translocated to the primary cilium of cells, so that it can be in close proximity with the Gli family of transcription factors Now, since it was known that the kinesin Kif3a was necessary for primary cilia formation, and moreover, that it was somehow, by unknown mechanisms, required for Smoothened function, and given that we had discovered that there was an interaction between barrestins and Kif3a, we wondered whether this interaction of barrestin and Kif3a might somehow be involved in the translocation of Smoothened, which was required, to the cilia, in order for it to function And in fact, we were able to find that when the system is stimulated by Sonic hedgehog, barrestin-2 is recruited to Smoothened, thereby bridging a ternary complex of Smoothened, barrestin and Kif3a The Kif3a motor then translocates the entire unit to the primary cilium, into proximity with Gli transcription factors, which can then be activated, leading to Smoothened signaling And in fact, we demonstrated, as has Marc Caron’s group, that in this case, barrestin-2 is required for Smoothened signaling Interestingly, GRK-2 appears to be required as well These interesting, and somewhat unexpected results, have raised the possibility that barrestins might function even more widely, as adapters to motors, to facilitate the cellular trafficking of other G-protein coupled receptors, and perhaps for other cargoes as well Now, the most recently appreciated function of the beta-arrestins, and one which I’ll be spending the most time on, is their ability to serve as signal transduction units in their own right And this was first discovered about a decade ago, for the non-receptor tyrosine kinase c-Src, but subsequently has been very widely studied for MAP kinases of various types, AKT, and a growing list of other signaling systems Again, ubiquitination of barrestins appears to be necessary for these functions My laboratory, in recent years, has been particularly interested in learning about other pathways which are activated through this mechanism and also, what pathways or consequences might lie downstream of activation of the pathways we already know about,

specifically the MAP kinases Even more remarkably, it turns out that the barrestins can serve as signaling adaptors, not just for G-protein coupled receptors, but it is turning out for specific examples of receptor tyrosine kinases, like the IGF receptor, cytokine receptors, ion channel receptors, such as the nicotinic acetylcholinergic receptor, and even Notch Now, in the course of studying the signal transduction properties of beta arrestins, we were led to an interesting discovery, which has turned out to have markedly facilitated this aspect of the work, while at the same time, serving to revise in significant ways some of our most fundamental understanding of how receptor biology works, and as you’ll see toward the end of the lecture, has actually led to a new approach to developing therapeutics I’m speaking here of our discovery of what we refer to as biased agonists Now, a biased agonist is a ligand which stabilizes a particular active conformation of a receptor, thus stimulating some responses, but not others. Seven transmembrane receptors, for example, can be biased toward a particular G-protein or beta arrestin. Mutated receptors can also be biased Now, I told you before, in the first lecture, that the classical, core concept, central dogma, if you will, of pharmacology, is that receptors can exist in inactive ‘R’ and an active state, R*, and that agonists, here displayed as ‘A’, stabilize or induce the activated conformation, with all signaling consequences then being a downstream result, or a function, of the concentration of active R* But the existence of biased agonists, which I’ve just described to you, immediately says that this is too simple That there must be multiple active states of the receptor, at minimum, for the purposes of this discussion, two One of which mediates G-protein signaling and the other which would mediate barrestin signaling And presumably there are a significantly larger number of active states than two But of course we can’t know how many at this point Let me illustrate this idea with some data about the very first biased agonist and biased receptor which we discovered And these both relate to the actions of angiotensin, the most potent vasoconstrictor which is made in the human body We had read about two reagents which seemed interesting to us based on our own previous work One was a mutated angiotensin octapeptide which is referred to as S-I-I angiotensin, for the 3 mutated residues in the polypeptide chain The second one was a mutated receptor, angiotensin receptor, shown here, which had mutations in two of the three highly conserved residues, which I mentioned to you in the first lecture, the so-called ‘dry’, D-R-Y, motif. Two of these are mutated to alanines and this is called the DRY -AAY receptor Now, the common feature of the mutant angiotensin peptide and the mutant angiotensin receptor is that they’re unable to activate G-protein signaling But, both can lead to internalization These interested us, because, as I’ve told you, we were aware of the fact that barrestins are able to serve as adapters, which mediate endocytosis of the receptors So, we obtained some of the peptide, had it synthesized, and we obtained the receptor mutant and we looked at their ability to activate G-protein signaling Now, what you can see here is that, in confirmation of results which had been previously published in the literature, neither the mutant receptor nor the mutant peptide, when applied to a wild-type receptor, could lead to G-protein signaling, as assessed in a classical second messenger generation So, here, we’re looking at PI turnover and when a wild-type angiotensin receptor stimulated with wild-type angiotensin, a classical dose-response curve is observed But absolutely no activity, for either the mutant peptide, applied to a wild-type receptor, or a mutant receptor stimulated with wild-type angiotensin

We also found no calcium release, no GTP-gammaS binding to G-proteins, so clearly, as reported, the mutant peptide and mutant receptor are devoid of G-protein stimulating activity However, both are very robust in their ability to recruit beta-arrestins Now, shown here are confocal micrographs. In this upper panel are shown HEK cells, which express the angiotensin receptor and which are also expressing GFP beta-arrestin 2 As you can see, the barrestin is diffusely expressed in the cytoplasm, but is excluded from the nucleus When such a cell is stimulated with wild-type angiotensin, twenty minutes later, the barrestin 2-GFP can be found concentrated in these endocytic vesicles That beta-arrestin had first been translocated to the plasma membrane, which we would have seen at an earlier time point, and has now been internalized. What you can’t see is that the receptors are also colocalized with it However, both the mutant peptide, when applied to a wild-type receptor, or the mutant receptor, stimulated with angiotensin, also robustly recruit barrestin and lead to its colocalization with the receptors in these endocytic vesicles This despite their complete inability to activate G-protein signaling So, clearly, these mutant peptide and receptor are devoid of G-protein stimulating activity, but do have robust ability to recruit beta-arrestin. Now, of course, the question becomes can they use that barrestin as a means of signaling into the cell? And to initially look at that, we chose a highly conserved signaling system which I’m sure is familiar to most of you, namely the MAP-kinase cascades Now, as you’ll recall, the MAP-kinase cascades are highly conserved from yeast to humans, they consist of three kinases in sequence: a MAP kinase, a MAP kinase kinase and MAP triple kinase The MAP kinases in turn are in several families, such as the ERKs, the JNKs and the p38 MAP kinases There are at least a dozen or so kinases at every level of this cascade So, there’s enormous complexity, many different types of G-protein coupled receptors activate these pathways, as do all sorts of other growth factor receptors, cytokines, et cetera And of course, the classical pathways that have been worked out involve phosphorylation by the MAP kinases, of transcription factors, leading to their activation, translocation to the nucleus, and regulation of all manner of transcriptional programs, shown here Now, a conundrum during the ’90s was, which was investigated, was the question of how, with all this complexity, with all these welter of enzymes, dozens of enzymes, how did the cell ever organize any specific pathway, such as, for example, RAF, MYC, ERK1, how did it ever organize such a pathway with any fidelity or reproducibility? This led to the idea that there might be scaffolding proteins which would bind together several members of a particular pathway and in yeast, a molecule, sterile-5, was discovered which appeared to function in this way We, in fact, were able to demonstrate that beta-arrestins, in particular beta-arrestin 2, was able to serve as a scaffold for several of these MAP kinase pathways For example, it was able to, it was able to scaffold a RAF-MYC-ERK pathway and an ASK-MKK4-JNK3 pathway So, it was able to serve as a scaffold for such pathways Let me show you one of the key experiments which led to these types of discoveries This shows that in terms of ERK MAP kinases, angiotensin gives robust activation of ERK MAP kinases in a cell which has been transfected to express the angiotensin receptor These are HEK cells However, the SII angiotensin, when applied to this cell, also leads to ERK MAP kinase activation, to the tune of about fifty percent of the wild-type and the same is true for the mutated angiotensin receptor

Now remember, in this case, there’s absolutely no G-protein activation and nonetheless, we’re getting half as much ERK activation, assessed by a simple assay of phospho- or activated ERK, we’re getting half as much ERK activity as we did with the wild-type system Now, if we use RNAi to knock down beta-arrestin 2, we lose about half the activity of the angiotensin stimulated system, but we completely ablate the activity of SII angiotensin or the mutated receptor, suggesting that they are activating the ERK system exclusively via beta-arrestins In the case of the wild-type system, if we block the G-protein arm with a PKC inhibitor, this activity goes to zero So, again, even though there’s no G-protein activity here, we are getting barrestin mediated activity So, to summarize many, many more experiments by my laboratory and other laboratories working now with dozens of different types of G-protein coupled receptors, have led to this type of understanding In the case of a GQ coupled receptor, such as the angiotensin receptor, it appears to be able to activate this specific effector system, ERK-MAP kinase, by two distinct, parallel and independent pathways One is the classical G-protein pathway, leading to second messenger generation, ERK activation and translocation to the nucleus to regulate transcriptional programs In the case of the barrestin pathway, in this case barrestin-2, this leads to activation of a distinct pool of ERK, which is sequestered and restrained exclusively in the cytosol and prevented from going to the nucleus This undoubtedly leads to the activation of distinct cytosolic substrates with distinct cellular physiological consequences We’ve been able to demonstrate this biology, as have other laboratories now, very analogous biology with quite a large number of other receptors And as I said, the mechanism appears to be a physical scaffolding of the different components of the ERK-MAP kinase Now, in our effort to understand the downstream consequences of this barrestin mediated signaling, in this case through ERK, but other pathways as well, we’ve recently been using a variety of techniques, some of them are shown here. We use these barrestin-biased ligands, which I’ve already mentioned to you, RNA interference, knock-out cells from barrestin-1 or barrestin-2 knock-out animals, the knock-out mice themselves and, as I will show you, a variety of proteomics techniques What I’d like to do is give you several examples, just examples, of novel pathways, barrestin mediated-pathways, which we’ve discovered just in the past several years So, for example, we have found a mechanism which operates downstream of the beta-1 adrenergic receptor in cardiomyocytes, or the angiotensin receptor in vascular smooth muscle cells It leads to transactivation of the EGF receptor and consequent activation of ERK, and it goes through a barrestin mediated activation of Src, then of metalloproteinases, which release the EGF receptor ligands, like HB-EGF This pathway appears to be cardio protective We found in vascular smooth muscle cells that through a barrestin mediated activation of ERK-MAP kinases, angiotensin is able to lead to ERK mediated phosphorylation and activation of a cytosolic kinase called Mnk, which phosphorylates and activates EIF-4E, a rate-limiting step of protein translation, leading to an increased rate of protein synthesis We found that in vascular smooth muscle cells, again, the angiotensin receptor, operating through barrestin-mediated signaling, is strongly anti-apoptotic And this anti-apoptotic activity is mediated through a concerted mechanism in which the barrestins activate ERK and PI3-kinase-AKT, two survival pathways, both of which lead to and converge on phosphorylation and inactivation of pro-apoptotic BAD Note that the ERK arm proceeds through phosphorylation of p90-RSK Finally, an interesting pathway, which I’ll say more about in a clinical context momentarily,

operates downstream of what was previously an orphan receptor, GPR109A, now known to be a receptor for the very important cholesterol-lowering drug niacin This pathway leads through beta-arrestin-1 to activation of PI3-kinase and AKT and then on to activation of phospholipase A2, generation of arachidonate, and vasodilatory prostaglandins And I’ll have more to say in just a few moments about the therapeutic relevance of this particular pathway So, this little cell I’ve constructed here, with each of the pathways I’ve just mentioned put into that cell, should begin to give you an idea of the rapidly growing complexity of these pathways and signaling networks, which operate downstream of barrestin Here are just a few pathways, delineated by my laboratory in the past several years If we were to graft onto this all of the currently known barrestin mediated signaling pathways it would be too complex to even look at And what this then has suggested is that we need some other way of looking at this issue of barrestin-mediated signaling Now, if we have any hopes of trying to understand the much broader universe of cellular signaling that’s mediated through barrestin, we really need to take a more general and global approach And so, that is the direction in which we have begun to move, because I think this type of work has clearly now suggested that signaling downstream of barrestins is undoubtedly probably every bit as complex and multi-faceted as that downstream of G-proteins And so in this regard, we’ve begun using a variety of global proteomics approaches and systems biology approaches So, in our first effort, Kunhong Xiao in our group conducted a proteomics screen to try to find out, or delineate, the universe of proteins bound to beta-arrestins either constitutively, or after stimulation of a G-protein coupled receptor. Again, the model we used in our initial experiments was the angiotensin receptor So, HEK cells expressing the angiotensin receptor were stimulated, or not, with angiotensin, and barrestins were immunoprecipitated and mass spectrometry analysis was used to define the proteins that were bound In addition to obtaining really most of the dozen or so known interactors that we had previously discovered on a case-by-case basis, Kunhong was able to discover dozens and dozens of novel interactors Perhaps not surprisingly, many of these fell into the category of signal transduction proteins Then of course we had motor proteins and several other groups Amongst the signal transduction proteins were a variety of protein kinases and phosphatases, trafficking proteins, small GTPases, et cetera. Many fascinating interactors which serve as springboards for discovery And then as I mentioned we found several families of motor proteins, including, of course, Kif3a, which I’ve already talked to you about Even more recently, we’ve begun doing quantitative global phosphorylation analysis of barrestin mediated signaling The procedure here is as follows We take a cell expressing a GPCR, such as for example the angiotensin receptor in vascular smooth muscle cells We then stimulate the cell with a barrestin biased ligand, such as SII-angiotensin Now, remember, this will activate only barrestin signaling, not G-protein signaling We can document that by then repeating the experiment after siRNA knock-down to eliminate barrestin mediated signaling We then use SILAC technology, stable isotope labeling of amino acids in cells, to quantify all the phosphorylation sites in the cell, before and after stimulation And then the phosphopeptides are characterized by tandem mass spectrometry Now, in some of our earliest results, again, using SII biased, arrestin-biased ligands stimulation of vascular smooth muscle cells, Kunhong was able to identify twenty five thousand individual phosphopeptides, derived from about 2,500 total phosphoproteins Now, not surprisingly, and in fact, gratifyingly, most of those don’t change when you stimulate with SII-angiotensin

You wouldn’t expect them to. In fact, about 85 percent of those phosphorylation sites do not change However, about 15 percent do. Ten percent go up and about five percent go down, after stimulation of barrestin-mediated signaling Some of the changes are very dramatic indeed But of course, the problem is, now that one has these huge piles of data, literally, these long lists of proteins and how much the phosphorylation is changing, the question is how does one begin to organize and analyze these vast quantities of data to understand what they’re trying to tell us about barrestin mediated signaling And so in this regard, we’ve been increasingly turning to the techniques of systems biology, using a variety of commercial packages for pathway analysis, as well as working with several collaborators, Avi Maayan at Mt. Sinai, and others at Duke, trying to understand and explore the best ways of analyzing this data I wanted to give you one specific example just to show you how this is working of a particular network which we have pulled out as being very enriched in the barrestin signaling universe. And this is a network for cytoskeletal rearrangement Now, cytoskeletal rearrangements are extraordinarily complex in terms of the biology and involve the concerted interaction literally of dozens of proteins Now, in this slide, and this is unpublished data, rather than listing the names of individual proteins, we’ve just given them numbers. But suffice it to say this is a reproduction with the names of the proteins removed of a currently understood signaling network which is involved in cytoskeletal rearrangements which occur, for example, in cell motility during chemotaxis Now, every protein colored red here was identified as being enriched in either the barrestin interactome or in the signaling phosphoproteome I just told you about or both So, clearly, many of these proteins involved in this cytoskeletal rearrangement network are being engaged, phosphorylated or dephosphorylated, in response to barrestin signaling Further, those which are circled in green here have already been validated in our laboratory on a case-by-case individual basis by techniques such as co-immunoprecipitation or others So these are very early days for us in terms of these types of approaches But hopefully what I’ve just shown you here can give you a sense of how we’re proceeding with this aspect of the endeavor Over the next few years, we plan to apply this type of analysis to several different receptors and several different cell types I also want to point out that exactly the same methodology and approaches can be used to explore barrestin mediated signaling which plays out, not just through phosphorylation, but through other types of post-translational modifications, such as, for example, nitrosylation or ubiquination Ok, so we’ve reviewed then, the interactions and the functioning of the barrestin and GRK system to mediate desensitization of the receptors, by several different concerted mechanisms, their ability to serve as adaptors for clathrin-mediated endocytosis and now this burgeoning area of signal transduction and scaffolding of signaling proteins But one of the most important implications of this rapidly growing area is that there are some important therapeutic implications And I want to begin to introduce that concept now And that’s shown in this cartoon The idea, as I’ve shown you, is that a seven transmembrane receptor can signal in two distinct ways: either through G-proteins or through barrestins However, in certain pathophysiological circumstances, G-protein mediated signaling can have deleterious consequences For example, stimulation of the angiotensin receptor, physiologically, through G-proteins, can raise blood pressure. Not a good thing in a patient with heart disease Similarly, beta adrenergic catecholamines, adrenaline, noradrenaline, working through the beta receptor,

can stimulate the force and rate of beating of the heart Not a good thing for a patient with effort-induced angina On the other hand, as I’ve showed you, some of the recently discovered signaling through beta-arrestins may have beneficial effects, such as, for example, an anti-apoptotic action Now, let’s talk about angiotensin receptor blockers, or ARBs Angiotensin receptor blockers are some of the most useful drugs, and certainly the most widespread, used drugs in cardiovascular medicine today Their utility specifically relates, as does that of beta-blockers, to their ability to block potentially deleterious G-protein mediated effects, in the case of ARBs, angiotensin, for example, though, to raise blood pressure On the other hand, ARBs block not only these deleterious G-protein effects, but they also block all effects mediated through barrestin, effects which 1) weren’t even known until the last few years, and 2), which, as I told you, may be beneficial So we hypothesized that perhaps a drug like SII angiotensin, which will block G-protein signaling, which it does quite effectively because it occupies the binding site and doesn’t signal through G-proteins, but which unlike conventional ARBs, is able to also stimulate barrestin-mediated signaling, rather than blocking it, which is what would happen with a conventional blocker, we speculated that this might represent a new type of drug And in fact, it turns out that SII angiotensin is able to slow the progression of heart failure in various animal models of heart failure, it lowers blood pressure and is, in fact, anti-apoptotic In a very analogous fashion, we have screened almost twenty beta-blockers, representing almost all beta-adrenergic blockers used in the world today to treat heart disease And the question we asked was, were any of these blockers able to actually signal through barrestins while blocking G-protein activation? Only a single one has showed this capability and that is Carvedilol, or Coreg, which in addition to its ability to potently block G-protein signaling, was able to weakly stimulate through barrestins In fact, carvedilol has unique efficacy in treating congestive heart failure, is anti-apoptotic and cardioprotective Another interesting example of a barrestin-biased ligand is shown here for the parathyroid receptor This particular PTH analogue has been known for years as a powerful competitive antagonist of parathyroid hormone In fact, it’s an inverse agonist, it actually lowers the activity, basally, of the parathyroid hormone receptor Now, you’ll recall that parathyroid hormone is a key regulator of calcium metabolism and a key stimulus for anabolic bone formation In fact, it is used clinically to treat osteoporosis Well, several years ago, Diane Gesty-Palmer in our laboratory discovered that this analogue, while it is in fact an antagonist of G-protein activation through the parathyroid hormone receptor, is actually a barrestin biased ligand which is able to activate, for example in cells, like osteocytes, is able to activate ERK-MAP kinase activity through the PTH receptor Recently, quite remarkably, she found that when this analogue was administered to mice, it led to anabolic bone formation, even though it can’t activate G-proteins at all And remember, the textbook explanation for the activity of parathyroid hormone to build bone is that this is exclusively a GS and cyclic-AMP mediated process Well, she found that administration of this ligand, which actually blocks G-protein activation, but which stimulates barrestin-mediated signaling, leads to anabolic bone formation without bone resorption Moreover, this effect was completely lost in barrestin-2 knock-out mice So, this barrestin-biased ligand for the PTH receptor may represent a new therapy for osteoporosis Now, biased ligands don’t need to be biased toward barrestin to have potential therapeutic utility

There are currently several, very provocative examples, where a G-protein biased ligand might well serve novel therapeutic purposes Let me give you a couple of examples I mentioned before that niacin, vitamin B3, administered in therapeutic doses, like several grams a day, is one of the most effective, probably the most effective agent we currently have available to us, for raising good HDL cholesterol and lowering triglycerides It also more weakly lowers LDL, but is not as effective in that regard as statins The problem with this potentially wonderful cholesterol modifying drug, niacin, is that very few patients can tolerate it because it leads to severe, severe, painful flushing, which is a result of the release of vasodilatory prostaglandins I also mentioned to you in the first lecture, and earlier, that the biological effects of niacin are mediated through this previously orphan receptor, seven TM receptor, GPR109A In fact, in knock-out mice in which this receptor is deleted, both the beneficial therapeutic effects on lipids, as well as the very annoying flushing, are both lost Well, in our lab, Rob Walters recently was able to show that the lipid modifying effects of niacin are actually mediated through G-protein activation, whereas the flushing, which is basically quantitated in mice by using a laser Doppler apparatus to monitor vasodilation in the ear lobe, he demonstrated that that effect is mediated through beta-arrestin 1 signaling, and not through the G-proteins In particular, it utilizes the pathway that I showed you before, which leads from GPR109 through barrestin 1 mediated activation of phospholipase A2, arachadonate and vasodilatory prostaglandin release In fact, the vasodilatory response to niacin was largely ablated in beta-arrestin 1 knock-out mice, although the lipid modifying effects, the desired therapeutic effect, is retained This suggests, then, that a G-protein biased ligand, niacin analogue for GPR109A might represent a novel and very effective form of therapy for cholesterol problems which would not lead to the very distressing flushing and which could then be tolerated by many more patients Now, another example of a potentially useful G-protein biased ligand would be one for the opiate, mu opioid receptor Now, of course, we all know that opioids are the single most powerful antinociceptive, analgesic, pain-relieving medicines that we have However, they cause a number of distressing side-effects which limit their utility Most obvious is tolerance, which requires more and more drug to get the same effect, but also side effects such as constipation, respiratory depression Now, the desired antinociceptive effects of opioids are mediated through stimulation of the GI proteins through the mu opioid receptor But recently Laura Vaughn showed that tolerance to opiates is markedly diminished in barrestin-2 knockout animals, barrestin-2 interaction with the receptor, causing desensitization, appears to be the key mechanism of the tolerance, so that’s lost in barrestin-2 knockout animals, and the constipating and respiratory depressing effect of opiates are also markedly depressed in barrestin-2 knockout animals So, this, then suggests that the desired therapeutic effects of the opiates go through the G-proteins arm, whereas the tolerance and other side effects go through the barrestin-2 arm And this, then, leads to the supposition that a G-protein biased ligand for the mu opioid receptor would be a particularly useful agent in treating pain, and one which would represent a significant advance over therapies that are currently in hand So, to summarize then, these several examples of barrestin-biased ligands, and G-protein biased ligands, should give you a sense of how this newly acquired knowledge of these distinct signaling pathways can be leveraged for therapeutic gain Now, the last topic I want to touch on has to do with trying to understand some of the biophysical basis

for these distinct G-protein and barrestin biased signaling mechanisms Now, from what I’ve told you before, it should be clear then that receptors, such as the angiotensin receptor or other seven-membrane spanning receptors, can exist in at least two conformations, probably more, shown by these differently shaped squiggles on the C-tail of the receptors Now, one of these conformations, presumably, leads to G-protein activation, whereas the other, leads to, for example, barrestin mediated signaling An unbiased ligand, such as angiotensin, appears to be able to do both, whereas a biased ligand, such as SII, can only do one, the barrestin or the G-protein So, we wondered whether barrestin biased ligands, I’m sorry, whether barrestins themselves might be able to also assume distinct conformations, conformations which would correspond to the distinct conformations of the receptor and which would then lead to distinct functional outcomes And so, to test this idea, we’ve recently been using a very interesting probe which is shown in this animated slide right here Now, as you can see, this is an intramolecular beta-arrestin-2 BRET biosensor This has been genetically modified, it was originally developed in Michel Bouvier’s laboratory in Montreal At the amino terminus is Renilla luciferase and at the C-terminus is YFP Now, when substrate coelenterazine is added, bioluminescence is generated by the luciferase, which, if in the correct orientation and close enough, usually said to be less than 100 angstroms, is picked up, the energy picked up by YFP and emitted as yellow light Conformational changes induced in the barrestin biosensor will modify the distance and orientation such that the BRET ratio will change In our initial experiments, we stimulated cells through the angiotensin receptor and looked at how that was reported by the barrestin-BRET biosensor So, in the initial set of experiments, we had the angiotensin receptor, stimulated by the unbiased ligand angiotensin When we did this, we observed an increase in the BRET signaling, presumably representing the two ends of the molecule being closer together, or in a more favorable orientation. In contrast, when we stimulated with the barrestin biased ligand, SII, we observed a decrease in the BRET ratio Both of these effects could be blocked by the competitive angiotensin receptor blocker Valsartan Then, we used the barrestin biased receptor, that angiotensin receptor DRY-AAY, which I told you about before Now you can see that with the barrestin biased receptor both angiotensin and SII led to a decrease So, these, and again, both were blocked by Valsartan, so these directionally opposite changes in the BRET ratio by the biased and unbiased ligand clearly indicate that must be inducing distinct conformations in barrestin Conformations which may well be associated with their distinct biological profiles We have observed similar results now with probably half a dozen different receptors, using both biased ligands and biased receptors Here’s an example for the PTH receptor I told you about that barrestin biased ligand before And with the parathyroid hormone receptor, stimulation by the unbiased PTH leads to an increase in BRET ratio, whereas stimulation with the biased PTH ligand leads to a decrease So, again, the variations are similar to what we observed with the angiotensin receptor So, this animated cartoon, with which I will conclude, demonstrates these distinct conformations of beta-arrestins, which correspond to the distinct conformations of the receptor, shown by the differing shapes of the receptor

A biased ligand, such as SII, leads to a conformation of the beta-arrestin in which the two ends of the molecule appear to be moving apart, thereby leading to the decrease in BRET ratio Whereas shown in the upper panel, when an unbiased ligand is used, we now get an increase in BRET ratio, corresponding to a moving together of the two ends of the molecule So, clearly the biased and unbiased ligands are leading to distinct conformations of the receptor and, this would suggest, the beta arrestin Now, going forward, the work in many laboratories, around the world, is now focused on trying to use methods such as X-ray crystallography and other biophysical techniques to document the detailed interactions and conformational changes which are characteristic of the distinct functions of barrestins and the receptors And as I mentioned to you before, this is a big interest in the case of receptor structural biologists as well, who are now interested in learning about the details, the conformational details, of those receptor conformations which lead to G-proteins as barrestin, or barrestin mediated signaling And hopefully in a few years, I’ll be able to stand before you again to present the results of such experiments