 I do want to introduce to you our keynote speaker. He is Dr. Robert Lefkowitz. He is the James B. Duke Professor of Medicine at Duke University. He is the first faculty member at Duke to be awarded a Nobel Prize while he was employed on the faculty at Duke. And there have been some very illustrious faculty members at Duke. This is quite an honor. And you probably won't believe this once you see him and you hear him. He said he's been at Duke for 40 years. I'm not going to presume to tell you any more about him. Kind of wouldn't make a whole lot of sense, I think. Please welcome Dr. Robert Lefkowitz. It's indeed a pleasure and an honor for me to have the opportunity to address you today. I'm going to give you two different types of talks back to back. And I, myself, don't know how long I'll spend on each of them. I'll sort of find out as you do. The first thing I want to talk to you about is a little bit about the career path that I followed, which may be of some interest to you. And you can see that I've titled these remarks a reluctant scientist. And as soon you'll understand what I meant by that. And then more toward the end of the presentation, I will tell you a little bit about the science that I've done. And then I guess we're going to leave 10, 15 minutes for questions at the end. And we want to wrap up by when? 4.30. OK. So I was born and raised in the Bronx. I have lived in North Carolina in Durham for 40 years since I joined the faculty here in 73. As you listen to me, you may say, wow, if his New York accent is this bad now, what was it like 40 years ago? And I think it was even more prominent. Anyway, I was an only child. And I went to public schools throughout my elementary and secondary education. One of the most formative experiences that I had. And I'll try to underscore where I think there are kind of life lessons and perhaps in my story. One of the most important influences on my entire career was my family physician in the Bronx, Dr. Joseph Fibush. He was a practicing general physician. And this in an age when physicians actually made house calls. And he would come to my house if I had a stomach ache or a fever, do whatever he did, lay on hands, write a prescription in an illegible hand. And wow, that just seemed amazing to me. And very early on, he became my role model. It seemed to me that there was really nothing higher one could aspire to than to be a physician. And I think I liked several things about the idea. One, it seems not that I understood much about science in grade school, but it seemed like he knew all the scientific stuff. In fact, beyond that, he knew all this stuff, these kinds of things that general folks, laypeople didn't know. And he was able to utilize this knowledge to heal and to relieve suffering and pain, et cetera. And I got it into my head early on that this was probably the noblest calling that one could have. It seemed to me much like the priesthood. And so from, I would say, the third grade on, I never really entertained any other possibility than that I would become a physician. And that became my single-minded career focus. Now, there are several things about that that I think are worth underscoring. One would be the importance of role models. And I think for the adults and teachers in the group, I don't need to tell you how central the exposure of the students to you really is. Because I think for most individuals who go into science or medicine, almost everybody will recount the importance of a scientific mentor or a role model for them. I was pleasantly surprised to learn, while I was in Stockholm last year, that my former student, Brian Kobilke, with whom I shared the Nobel Prize this past year, that he too, he was a physician as I am, that his most important role model as a child growing up was also his family physician. And yet, as you know, we both ultimately gravitated into careers in science. So the first point is the importance of role models. The second is the notion of a calling. I don't know if you all understand what I mean by a calling, but a calling, is something which we generally associate with the clergy. But it's a special sense, experienced at a deep emotional level, not so much even at a cognitive level, that you're supposed to do something, hopefully something of some value or virtue in its own right. And I would have to say that even as a youngster, I experienced the notion of going into medicine as a calling. Anyway, I stuck with that idea and found myself in my early teens at the Bronx High School of Science. I don't know how many of you have heard of the Bronx High School of Science. To me, your school is very much patterned after the Bronx High School of Science. It was a public school. It was not a residential school. It's now 75 years old. And admission to that school was open to anybody in the city of New York. But you have to take a competitive examination. And to this day, the only criterion for admission to that school, and this has become somewhat controversial, I gather, from what I read, was how you scored on this competitive examination. And you had to be in the top, I don't know what, but it was the very highest scores. So I went to the Bronx High School of Science with a lot of very, very smart kids. And I was in the class of 59. We all, of course, went to college. Almost all of us went to graduate school or medical school. It was a remarkable experience. We had the opportunity to be amongst the first classes to take AP courses, which I assume you all take. And I took quite a number of those. And when I went off to Columbia College in New York City to get my bachelor's degree, I was awarded 20 credits out of the 120 that you needed to graduate for the coursework I had done in AP courses. Because I had 20 credits and was given advanced placement, I was actually able to graduate from college in three years. And at the time, I was anxious to do that because I just couldn't wait to get to medical school. And I went to medical school in Columbia, so my education is playing out all in New York City. I went to Columbia Medical School, the College of Physicians and Surgeons. And those were some of the happiest days of my educational experience. I had been waiting, as I've told you since probably age eight or 10, to learn how to become a physician and to learn that secret stuff that only doctors know. And here I was, and I was learning it. And I loved every minute of it. And I did my house staff training in internship and residency at Columbia. Now, I would point out that I had no interest whatsoever at any stage in my career up to then of doing research. It never entered my mind that I would be a scientist. In fact, we had several options to do research while I was in medical school. There were blocks of two and three months that you could spend either doing clinical rotations on awards or doing research. And on every occasion, I opted for the clinical electives. I had no interest in research whatsoever. I was very interested in science. I was a chemistry and biochemistry major in college. I found it fascinating. But I wanted to use that scientific knowledge directly at the bedside of sick patients. I was not interested in going into a laboratory as a way of contributing to medical progress. Now, where that began to change was in 1968. I graduated from medical school in 66 and did two years of house staff training. But in the late 60s, something called the Vietnam War was raging. And this was a very unpopular war. And in something which may seem very, very foreign to you students, we had a draft, a military draft. And we had a separate draft for physicians, a doctor draft. So you had to go into the service. The only question was whether you would be in the army or the Navy or the Air Force. And you would spend one of your two years of required service in Vietnam. As I said, it was a very unpopular war. And many of us were not anxious to participate. There weren't a lot of options to fulfill your military obligation. And at the same time, not serve in the armed forces. One of the very few was to secure, and it was very competitive to do so, to secure a commission in the United States Public Health Service. The United States Public Health Service at the time was considered not just one of the uniformed services, but was considered one of the military services. And they oversaw, for example, the Coast Guard. They also staffed the National Institutes of Health, the CDC, the Center for Communicable Diseases and several research installations in the far Southwest which did research on Native American Indian populations. Well, I was able to secure a commission in the Public Health Service and was assigned to the NIH where I began my research career in 1968. And I can tell you, it was not pretty. The first 12 to 18 months of my 24 month assignment there were without any discernible success or forward progress on my project. I was an utter and abject failure. And I think that's another important thing to take away. If you're going to do scientific research, failure will be your handmade. You will fail much more than you will succeed. I remember one of my mentors saying to me during this dark period, he said, Bob, do you know the difference between a highly successful scientist and one who's not successful? And I said, no. He said, well, for the unsuccessful scientist, maybe 1% of what they're trying to do will work. But for the real superstar, it might be as high as two, or 2.5%, and boy, was he right. In fact, I think he was over-generous. So you have to learn that if you are gonna pursue a career in research, you have to be ready to deal over and over and over with failure. Of course, how you define failure is also an issue which we can talk about because if you're shrewd, every failure teaches you something so that you're a little smarter for the next experiment. Well, I was pretty miserable with the research. And since I had always wanted to be a physician anyway, a practicing physician, this really sort of steered me away from any nascent interest I might have had in research. And in particular, I had a very personal experience about six months into my time at the NIH. It was Thanksgiving time, the NIH is in Bethesda, Maryland, the suburb of Washington. And I decided to travel home with my young family to New York City to spend the Thanksgiving holidays with my parents. I was very close to my father, I was an only child. He was an accountant, knew nothing about the specifics of what I did, but had always been a wonderful advisor on all matters. And so I had a long talk with him and I told him how unhappy I was with my repeated failures during those first six months. And this was a new experience for me. I was always top of my class. I had never failed at anything that I'd ever tried to do. And here I was meeting with no success whatsoever. Well, you know, for my parents, their dream had always been for their only son to become a physician. And he counseled me very reasonably. He said, look, you always wanted to be a doctor. You know, you'll fulfill your military requirement there at the NIH, just get through it. And then you'll go back to your clinical training and go on with your career as a cardiologist. I was already interested in cardiology just as you had always hoped and planned. Well, that made a lot of sense to me. And I felt very relieved. And I traveled back right after the Thanksgiving holidays to the NIH to continue my work. And it began immediately applying for residencies and fellowships to follow my two-year stint at the NIH. Well, it turned out that was the last time I ever spoke with my dad. He died suddenly of a heart attack two weeks later in mid-December. And that deeply affected me in several ways. But one of the strangest ones was that I almost felt I had made, if you will, a pact with him. We had a plan. I was gonna pursue my clinical training and that's where I would go. Well, things often don't go the way you anticipate. And toward the end of my two years at the NIH, things really began to break for me. And I met with my first successes and published several what turned out in retrospect to be important papers. But by then I had already committed myself to further clinical training in Boston at the Massachusetts General Hospital, which is one of the main teaching hospitals of Harvard. And even though I was under a lot of pressure to stay at the NIH, I was not gonna break my commitment. And so off I went. And then I had another really important experience. During the first six months back at clinical work, I loved it, I enjoyed it very much. But of course I was doing no research. And the importance of this experience was that I realized just how much I missed the stimulation of being in the laboratory, of having data every day, of being able to grapple with it and analyze it. And I began to realize, you know, this is gonna be tough if I spend the rest of my career and I don't have any data anymore. And so during my second six months of my senior residency and against hospital regulations, which mandated that although we had six months of elective time, it all had to be spent doing clinical work because we were paid by clinical patient derived revenues. I basically surreptitiously invagaled myself into the laboratory of a scientist and spent the next six months doing basic research. They eventually caught me one night. The residency director was coming through the halls and found me with a racket test tubes. But they just slapped me on the wrist and kinda looked the other way. I went on to spend two more years there doing research and completing my clinical training in cardiovascular diseases. And then in 1973, I moved to Duke to join the faculty just 40 years ago. When I first came here, my research was beginning, I didn't accomplish all that much surprisingly during the residency years and in Boston in research, but it certainly kept my drive alive. And when I came to Duke, I was still kinda undecided as to which way my career was going. I would say the first year or two, I probably spent 50 or 60% of my professional time doing research in a new laboratory that I was setting up and maybe 40 or 50% doing clinical work attending cardiology clinics and making rounds. And by the way, I continued making rounds. The teaching rounds for 30 years until 10 years ago when I turned 60. But then within the first couple of years, things really started to take off. And I got deeper and deeper and deeper into the research. And I found within about three years that what I was dreaming about on the way home in the shower was not the patients that I saw in clinic. It was my experiments and how I could get them to work, how I could move the projects forward. And so over a period of time, I began to evolve more and more into a fuller time scientist. But you know, I mentioned reluctant scientist. One of the things I realized in retrospect that held me back was, you know, I had this deal with my dad. I was gonna be the cardiologist that the family had always dreamed of. And here I just felt this impulse to be in the laboratory almost like in some way, I was being disloyal to his memory. Well, of course, I eventually came to my senses and just allowed it to happen. And the remarkably fortunate thing for me, of course, was that I was feeling a calling again, but it was for the second time. Now I felt called to research. And again, I think the lesson in both these experiences that I had is that when it comes to figuring out what you're gonna do, you can't figure it out with your head. You've gotta figure it out with your heart. You just gotta listen to your own inner voice in terms of what is it you wanna do? What is it you're good at? What kind of work could you do that actually feels like play? If you can figure that out and just sort of listen to what you're telling yourself, I think you'll have a really good chance of being successful. And for sure, you'll be a lot happier. So I eventually evolved further and further into a very basic or fundamental scientist doing biochemistry and chemistry. My dad never got to see any of it. I would say 25 years ago, I started getting awards for my research. I know he would have been extremely proud of it. My mother was proud, but I don't think she ever fully came to grips with it either. She would, every time I would talk to her, it seemed, and she called me Bobby, she would say, Bobby, have you figured it out yet? So what she meant is that her notion was that I had basically been, I don't know what the right word is, seduced by some crazy scientific question. And that if I could just figure out the answer, I'd come to my senses and return to a career as a physician. Well, alas, for poor mom, it never happened. And at the time of HUD death, of course I still haven't quite figured it out. And I haven't figured it out yet. Anyway, things have gone well for me in recent years. I've enjoyed my career to date at Duke quite a bit. Quite a bit, as I mentioned, I continue to function as an academic physician, teaching students and fellows on the wards until 10 years ago. Now all my teaching is confined to the laboratory where I remain as active as I've always been. As you heard, I will turn 70 next month, month from today. And the word retirement doesn't really mean anything to me at this point in my life. And I very much enjoy what I'm doing. It's interesting to me that my co-nobel laureate, Brian Kobilka, who was my fellow in the mid-80s, is like myself, a physician and a cardiologist. He served in my laboratory for four or five years in the 80s as a cardiology fellow doing research, much as I had a generation before up in Boston. And yet, where it brought us to this research was the Nobel Prize in chemistry, which has bemused some individuals who think of themselves as real chemists. People often ask me, were you surprised by winning the Nobel Prize? And the answer is yes and no. No, I wasn't surprised in the sense that for a number of years individuals have told me that, hey, Bob, you may win the prize. You're gonna win the prize, et cetera. It didn't happen until last year. So in that sense, yeah, had it ever entered my mind, sure. But would I have ever expected it would be in chemistry and not in medicine? No, at least not until very, very recently. And in particular, on the day that I received the call from Stockholm at 5 a.m. East Coast time, I was totally shocked because I had heard no inklings, no rumors, nothing to alert me that this might be in the works, much less that it might be in the works in chemistry. You may or may not know that the Nobel Prizes are announced in a specific sequence each year. Mondays medicine, Tuesdays physics, Wednesdays chemistry, and Thursday and Friday you have economics, literature, et cetera, and pieces the following Monday. So Monday had come and gone. I certainly hadn't gotten any calls. Not that I was expecting one. Wednesday morning is the chemistry announcement and you can be sure that I was sleeping very soundly when that call came in. Fortunately, my wife picked up the phone. I would not have heard it. So yes, it was surprised, but in a certain overriding sense, maybe I thought it might happen someday. And I can assure you this, that the idea that I shared this prize with somebody who had trained in my laboratory, someone whose career I know I had shaped in a fundamental way, and someone with whom I had kept up and continued to advise and talk with over the years was a very, very special treat and one which just sort of amplified the significance of the whole thing in my own mind. So that's how I got here. That's how I come to be standing in front of you. Let's talk a little bit about why I got the Nobel Prize. So I got the prize with Brian for elucidating the characteristics of cellular receptors. So what are receptors for hormones and drugs? Well, I don't know if you can read that, but a receptor, here's some definitions for you, is a molecule on or in a cell with which a drug, hormone, neurotransmitter initially interacts. Ligand, okay? A ligand is a compound which interacts specifically with a receptor. An agonist is a ligand which stimulates a receptor. An antagonist is a ligand which binds to but does not stimulate a receptor, synonym, a blocker. So if you look at the left panel here in this little animation, here's a receptor will come in a moment more to the idea of why I've drawn it like that. Here's a drug and it's an agonist. It stimulates something to happen. Here are some examples of agonists. Adrenaline or morphine. Both are drugs used in clinical medicine. Here's an antagonist. It binds to the receptor just like the agonist does, but doesn't do anything. Notice the receptor doesn't change, nothing happens. It just sits there. But because it's sitting there, the agonist can't sit there. That's a blocker. I guarantee you there are enough adults sitting in this room that there are people sitting in the room who are taking some form of a blocker, whether it's a beta blocker or something called an angiotensin receptor blocker or others. Antagonists and agonists are used in clinical medicine all the time. So let's give you a sense of how this fits into normal physiology. Because of course, receptors like all molecules in our body evolved to deal with real life situations, not with synthetic drugs that we make. So what's the example I can give you of how receptors might function physiologically? Well, I have used throughout my career a particular kind of receptor. It's an adrenergic receptor. The term is derived from adrenaline, adrenergic. There's one particular kind of adrenergic receptor called the beta-adrenergic receptor. We'll talk about that in a minute. And I've used it as a model for most of my work. The beta-adrenergic receptors are the target of a class of drugs called beta-adrenergic receptor blockers, shorthand for which is beta blockers, okay, which are some of the most highly used drugs in the world for the treatment of coronary artery disease, angina, hypertension, anxiety, a wide variety of things. Now, adrenaline and the adrenergic receptors are crucial in what's generally referred to as the fight or flight response. So for example, let's say a stimulus, a frightening stimulus is received by the brain. Immediately a hormone or a neurotransmitter, for example, adrenaline, synonym is epinephrine, is released into the bloodstream. And that adrenaline molecule shown right here binds to receptor in the plasma membrane, in the outer surface of the cell. And something happens, you get a signal which goes to the interior of the cell and does stuff and makes the cell do something. If that were a heart cell, it would make it beat faster and stronger. If it was a muscle cell in the airways, it would make them dilate, okay? We often refer to these molecules that are released into the cell as second messengers. And the first messengers would be the molecules like adrenaline. The adrenaline can't get across the cell membrane, so it stimulates a receptor in the surface of the cell, changes that receptor in some way, and now you get second messengers generated inside the cell. Many years ago, 50 or 60 years ago, a very famous scientist, a pharmacologist named Raymond Alquist, was able to use a variety of drugs that were available, adrenaline and noradrenaline, which are both drugs and hormones and neurotransmitters, as well as several chemically related molecules to define what he called alpha adrenergic or beta adrenergic receptors. This was simply based on observations where if he took maybe half a dozen of these compounds, adrenaline, noradrenaline, and three or four others, and looked at their ability to stimulate things like how fast the heart beats, how much in a tissue bath, a blood vessel is contracted, the extent to which an isolated preparation of a gland like a pirated gland, a salivary gland, secretes something in response to these drugs. So he looked at the ability of these half a dozen drugs to stimulate these responses. And when he did that, he found that he got one of two patterns, either drug one was better than two, is better than three, is better than four, was the other way around, but it was always one of those two patterns. He said, well, maybe there are two different receptors which have a different specificity. He called these alpha and beta receptors. Nonetheless, he did that work in 1948 and was rewarded with the Lasker Prize, which is a very prestigious prize in medical research. And nonetheless, the idea of receptors in his day was very different than it is today. And it was kind of mystical and it certainly wasn't anything chemical or biochemical. In fact, as late as 1973, now you may recognize that as the year I came to do, Alkwist wrote the following in a journal called Perspectives of Biology and Medicine. And he wrote this after appearing on a symposium program with me in which I presented some of my very, very earliest work. And as you can see, you wouldn't know that impressed. He wrote the following. This would be true if I was so presumptuous as to believe that alpha and beta receptors really did exist. This from the man who came up with the idea in the first place. There are those that think so and even propose to describe their intimate structure. To me, they're an abstract concept conceived to explain observed responses of tissues produced by chemicals of various structure. So he didn't really believe in these receptors as real molecules. And in fact, when I came to Duke in 1973, this was the general, shall we say, state of play in the field. There was no real hard evidence that there was such a thing as a receptor. So when I came to Duke and began my research program 40 years ago, I set as my first goal to try to develop a way of directly studying their receptors, of measuring them, characterizing them, so that I could begin to isolate them, prove that they existed and find out how, you know, what they were and how they functioned. And so I basically used a very simple approach and those are often the best in which I would take molecules, the simplicity I've shown you, the adrenaline structure here, and just radioactively label them and try to see if I could get the molecule to stick to the receptor and actually measure that. I was able to do that and as a result, we were able to do all kinds of things. And one of them was we were able to use these radioactively labeled tags for the receptor to begin the difficult job of trying to isolate them. Now, receptor isolation was extraordinarily difficult. These receptors, and again, we still hadn't proved that they even existed, are very rare. They're essentially almost trace contaminant proteins in the plasma membrane. So if you took all proteins out of the plasma membrane and laid them out, and just at random took 100,000 proteins out of a piece of plasma membrane from a cell, one would be this receptor. 99,999 are other things, sometimes multiple copies of very abundant proteins, but they're almost none of these. So how are we gonna do it? Well, first of all, the receptor is stuck in the plasma membrane. I mean, it's a solid thing. You can't, like a film, like when you blow these thin films of soap that we all used to do. So you can think of the plasma membrane that way in which proteins are inserted. So first thing we had to do was solubilize the receptors. Now, this is just an animation. We didn't use magnets, they're not metallic. But the idea is we had to get the receptor out of the plasma membrane in a soluble form. And then once we did that, we had to somehow purify it. This work, not just for the beta receptor, but for other, they're actually at the time of four different subtypes that we thought there might be alpha and beta and subtypes as those, took myself, my students and fellows, 10 years. And it was extraordinarily difficult. And whatever frustration or difficulty I might've thought I experienced back in the NIH for those 12 or 18 months was nothing compared to this. But we succeeded. And the key to our success was the use of a technique called affinity chromatography. So what we did, and this is painstaking work, we figured out how to take drugs, which would say beta blockers or alpha-adrenergic receptor blockers. And we covalently, chemically linked them through various chemistries to solid supports like agarose beads. We then took our solubilized receptor preparations. Again, we would use detergents, soaps, if you will. And that, just finding one that would work as to say we take the receptor out of the membrane and not destroy its characteristics. We would take these soluble receptors and pass them over columns of this material. The receptors would stick to the drugs. I mean, that's what they do for a living, right? They bind these drugs. They would stick. We could then wash the columns, wash away everything else, and then elute the receptors back off by putting in a high concentration of another similar drug. So we would call this biospecific adsorption of the molecules to the column and biospecific elution. And we developed different affinity chromatography procedures for each of the several types of receptors. We coupled those with more conventional forms of protein chromatography and were able to ultimately isolate each of the receptor types. And I said this was 10 to 12 years of work. These are so-called SDS polyacrylamide gels where you can run protein solutions and proteins will separate according to their molecular weights in terms of how far they migrate in the electric field. And as you can see, there's only one protein in each lane. In the starting material, the lanes would be black. There would be thousands of proteins. We had to obtain approximately a 100 to 300,000-fold purification. And we had their micrograms of material when we were done. But the material that we had would bind those radioactive ligands that I told you about in just the right way. We could look at the ability of other drugs to compete and knock the radioligand off the receptor and we would get exactly the right results. So we knew that what we were isolating as a protein had all the characteristics of the receptor. It would bind things in just the right way. The beta receptors would bind beta blockers, very tightly, but they wouldn't bind alpha blockers and vice versa. But receptors really do two things. They bind ligands, like adrenaline, or morphine, with a great deal of specificity. Generally, with stereospecificity, I'm sure you've learned about that in your chemistry and biology courses, that is to say one stereoisomer is much more potent and binds with much higher affinity than the mirror image stereoisomer. And there's a remarkable selectivity which suggests that the binding pocket of that receptor must have very specific geometric constraints. But the second thing that a receptor has to do is do something. As I showed you, it's got a trigger or response in the cell that leads to the generation of some second messenger or something else. And to prove conclusively to the scientific community that we had truly isolated the receptor, we had to show not only that they bound ligands with just the right specificity, but that they could do something. So how are we gonna do that? So here's my little magnet cartoon again. So now we've got the isolated molecule. So now we take a lipid vesicle, sort of like that lipid membrane we talked about. And we reinsert the receptor back into that. So now I've got vesicles with receptor. So now I need a cell maybe which doesn't have this receptor. Turns out that was hard to find. Turns out all mammalian cells have beta-adrenergic receptors. But we found the cell in an amphibian, Xenopus lavas, it's erythrocytes have all the enzymatic machinery to respond to beta receptors, but they don't have the receptors. In consequence, this cell doesn't respond to adrenaline. Here it goes, bounces right off because there's no receptor there to bind it and start the ball rolling. Let me just say that while this cartoon is up, G protein stands for GTP binding protein, or guanine nucleotide regulatory protein. This is the next protein in the chain that these receptors activate. So once a receptor binds an agonist and it's doing something, it does that something by interacting with the G protein, which is sort of a middleman, and that then interacts with an enzyme that makes a second messenger. You may have heard of cyclic AMP in your biology classes, which is a second messenger and there are others. So this cell, this erythrocyte from Xenopus lavas has some kind of receptors, doesn't have beta receptor, and it has these G proteins and the other machinery, but it can't interact with the adrenaline because there's no receptor. So then we took our lipid vesicles that are containing these receptors and we used polyethylene glycol to fuse it to the membrane of the cell, thus inserting, reinserting these isolated receptor molecules into the cell membrane, okay? And we could show using our radioligand binding techniques that this is what we had achieved. Now, bingo, the cells responded, which we could measure to adrenaline. So we had proved once and for all that the isolated single type of molecule that we had was truly the receptor. It bound ligands with all the right characteristics and it conveyed on a receptor-less cell the ability to respond to adrenaline. And this was really then the true proof that the receptors existed. Now, we were then able to, we had reached that point by the mid-1980s. As I said, there was about 12 years of work. The next step was to find out what they looked like. And this, by the mid-80s, molecular cloning techniques, which I'm sure you've been learning about, had been developed. And since we had isolated, frankly, no more than about 25 to 50 micrograms of any of these receptors, we were able to use a variety of techniques, enzymatic and chemical, to chop the receptor up into little pieces to isolate those peptide fragments on chromatographic columns. And use micro sequencing techniques, which were just being developed, to find out little stretches of sequence, of amino acid sequence of the receptor. We could then use those and the knowledge of the genetic code to design oligonucleotide probes, which we could then use to clone the gene and the CDNA. I'm sure you've learned hopefully a little bit about these techniques. So that we could deduce the entire amino acid sequence of the receptor. And when we did that, we made a remarkable discovery. So, first of all, the receptor, and this is the sequence of the beta-2-adrenergic receptor, which we've been working with. Each of those little balls is an amino acid. There are about 420 of them in this particular protein. There's a letter from the single letter code in each of those saying what amino acid is. You can't see them, it doesn't matter. We could tell from the deduced sequence that there were seven recurring stretches of very hydrophobic amino acids. That general of about 25 residues in length. This generally indicates a transmembrane-spanning domain that's inserted in the lipid bilayer. So here's one, here's one here, and there are seven. So we often call this a seven transmembrane receptor because it has seven transmembrane segments. What was most remarkable in this discovery is that the residues shown in blue were identical to another protein that had been sequenced only a year or two before. And that protein was redoxin, the visual pigment that we use to perceive photons of light. Now it was known at the time that redoxin, once it is triggered by a photon of light, stimulates a G-protein and leads to second messenger changes. So that's analogous with what I'm telling you. But nobody dreamed that redoxin would look like the beta-hydrenergic receptor or anything like it. So immediately we perceived that perhaps, and it also had seven membrane spans. We immediately said, oh my goodness, there were lots of other receptors known that were G-protein coupled. The list is very, very long. Dopamine, serotonin, glucagon, it goes on and on. We said, wow, maybe all of them look like this. Maybe there's a family of these seven transmembrane receptors and they're all, if you will, siblings. And in fact, very quickly, we were able to clone the genes for these other ones that we had purified. The very next one was something called the alpha-2. Very quickly we'd clone four and then eight different receptors, they all looked like this. Then others using our sequences and assuming that all the receptors would have at least some similarity in their sequences started cloning other receptors. And very quickly the family grew until by the 90s we knew that there were about a thousand different genes in this gene family encoding receptors for all manner of things. All of them have these seven transmembrane spanning sequences. All of them share very significant sequence similarity in their amino acid sequences. It slides a bit old of the 800 to 1,000 receptors of this type, different types. 200 of functionally known receptors. By that I mean, I can tell you it's a glucagon receptor or a serotonin receptor. But the majority are what we call orphans. So we know it's a receptor from its gene. Remember the human genome has been sequenced so we know the sequence of every gene. There are genes that encode things that look like this and they're expressed in certain cells. But nobody knows what the ligand is. These represent an extraordinary opportunity for drug discovery at the present time. Then we know that there are hundreds of sensory receptors. So of our senses, three of them are carried out by molecules that look just like this. Rudopsin, fibison, essentially it's a photon receptor. Smell and taste. Smell is the largest subfamily. There are probably in the human genome 300 or 400, about half of all the receptors are smell receptors. Accounting for why smell is such a nuanced thing. Of the various taste modalities, two work through these receptors, bitter and sweet. So the family is generally referred to as G protein coupled receptors or as seven transmembrane receptors. Of great importance is that, as it says on this slide, more than half of all prescription drug sales in the world today are of drugs which target these receptors directly or indirectly as either agonists, that would be true for say, adrenaline or morphine, something like that, dopamine. Or as antagonists, beta blockers, angiotensin receptor blockers, anti-histamines of any kind. There are two types of histamine receptors. They look just like this. They're members of this family. H1 receptors, those are the classic anti-histamines that you take when you have an allergy attack. In my day, you know, it's pyrobenzamine. Today, it's whatever you take. There are lots of them. H2 is a different type of histamine receptor. It blocks acid production in the stomach and blockers of those types of histamine receptors are of course sold over the counter now for the treatment of gastric hyper-city. So it goes on and on and then of course all the opiates, it's morphine. All the different opiates that are used for pain relief and many, many others. So the families of extreme importance and the types of techniques that I've been describing to you have been very, very useful and were adapted as we were developing them in the 70s and the 80s and the 90s and were rapidly leveraged by the pharmaceutical industry to very quickly be able to develop new drugs for patients to take. In the last few minutes, I wanna tell you about one other aspect of the research. And that has to do with a phenomenon which is not just pervasive, but is universal amongst all of these receptor mediated systems. It's called desensitization. And it refers to the phenomenon shown here for two different receptors, the beta-adrenergic receptor and the angiotensin receptor. And it's simply that when you stimulate them, either adrenaline here or angiotensin, angiotensin as I'm sure some of you know is an adrenal hormone from the adrenal cortex, it constricts vascular smooth muscle, raises blood pressure, blockers are used to treat high blood pressure. But when you stimulate the receptors, you get a signal, in this case, it's a second messenger cyclic AMP, in this case, it's a different second messenger called dais, a glycerol. But very quickly, within seconds, even in the presence, continued presence of the stimulus, you get desensitization, you lose the signal. Things shut down. And in the case of agonists, that's a very significant break on their therapeutic efficacy. So I've always been interested in how that happens. And in fact, over the years, we discovered the basic biochemical mechanism of that. Turns out when you stimulate these receptors with an agonist, they told you they interact with these G-proteins, you get second messengers in all kinds of signals. But very quickly, you also get desensitization. The desensitization is carried out by proteins from two other gene families, two families of proteins that we discovered in the 80s and 90s. One is a family of kinases. You may have learned that a kinase is an enzyme which transfers a phosphate group from ATP to a substrate. In this case, the substrate is the receptor, but even more specifically, the activated receptor. So GRK stands for G-protein-coupled receptor kinase. There are seven enzymes, this one's GRK2. Phosphorylates the receptor, usually on multiple sites on this carboxyterminal tail in the cytoplasm. And this leads to the binding of a second type of protein, which we discovered, called beta-arrestin, so named because it arrests or stops signaling. When the barrestin binds and the trigger for its binding likes to bind to the phosphates. So once the activated receptor is recognized by the kinase, it's phosphorylated, the beta-arrestin binds, and then that really blocks the G-protein from getting in there. And so you have desensitization. So just in cartoon form, here's the agonist, the receptor changes shape on the inside. Here's the G-protein being activated, whoops, here comes the GRK, it puts those phosphates on. That's actually what barrestin looks like, that's its crystal structure, and now it's in the way. So you have desensitization. Now, over the last 10 years, what my work has focused on is shown in this slide. We made the very surprising discovery that this system of a GRK and barrestin, which we had discovered over a period of many years, as the mechanism which turns off G-protein signaling, we found out that it actually can itself serve as a signaling system in parallel to the G-protein. In other words, it gives the receptors a different way to signal. This is a total surprise. And so really, this system is, if you will, bifunctional. On the one hand, it desensitizes the G-protein signal. But on the other hand, it sets up an entirely different way to signal to the inside of the cell. And I won't go into, it's very complicated how that works, but it's fascinating. So closing thoughts. I've had a magnificent time with these magnificent transmembrane receptors. And even when you're doing very basic science, as I've been doing, fundamental biochemistry, we're now doing x-ray crystallography, you can still generate new ideas for clinical medicine. And that, for me, has been one of the most gratifying parts of the kind of career that I've had having been originally trained as a physician. And I would tell you that you're only the second high school audience that I have spoken to since I won the Nobel Prize. The first one was when I was in Stockholm, I was invited to address a group much like this, not quite as large, in one of the leading high schools in Stockholm. And they have posters, which I guess you can order. They're very lovely in each of the scientific areas. And so I was at, so we have a couple in my lab, and I say I'm sure you can order them from the Nobel Foundation. They print up many of these. And so the students had the poster for the chemistry prize. And so I was doing a signing. I was standing off to the side after my lecture, signing their posters. And one young lady comes up to me, and then some guys come up, they want me to sign their arm. I said, well, okay, I'll sign their arm. But then this young lady comes up to me. Well, look at here. She asked me to sign her forehead. And that was an unusual request. I'm sorry I didn't get her name, because I'd like to know if she's washed that off yet. Because it was only a few months ago. The other thing I would remind you is that in this day and age, you don't do all this research and win a Nobel Prize standing by yourself at a lab bench working all alone. The work is done by a lot of people. This is a picture taken 10 years ago at my 60th birthday when a bunch of my alumni returned. I had trained over 200 people in my laboratory, in my career. We had about 100 of them back at this time. And this guy here by the way is Ko Bilka who shared the prize with me. So we were taking a photograph. I had been standing right here. And then all of a sudden they hoisted me up. But I liked the picture because it gives credit where the credit is done to these people. I always tell people, nothing is impossible for the man who doesn't have to do it for himself. And in fact, they deserve the credit and they're hoisting me up like this is in a sense very emblematic of what the whole thing is about. And then the final thing I'll just put on here, if I can find it, absent this with all the respect I suspect I would not have been invited here to see you. But in case if you've ever wondered what's it like when you receive the Nobel Prize, what goes on there? It happens in an amazing concert hall even more balconies than you've got here. And that's me and Kovilka. In White Tie and Tails, I would point out. And this is a member of the Nobel Prize Committee in Chemistry. This is Jim Watson. Anybody know that name, Watson and Crick? It's the 50th anniversary, it turns out, of his prize for the double helix. And he was back and there he is. And here's my big moment. The King and Queen behind them are the lovely young princesses. And he hands me the medal. And now they rehearsed us in the morning. I got about three times, once to the King, once to your colleagues, once to the audience. And this is my lovely wife, Lynn, who seems very proud. And that was my grandson next up. So thank you very much. Thank you.