 Thank you for the introduction and I regret that I can't be there in Omaha in person to Provide you with this discussion History is a Interesting thing and I'm going to try to take you back to in some cases to your childhood and in some cases to your pretty womb experience and The work that we're going to be covering starts in the Really in the 50s and kind of peaks out in the 60s and Courses continue down up until now But we want to try to talk about Receptors receptors particularly for the estrogen receptors for steroid hormones in general Probably will come up come to play in this talk But to try to take you back to the time where this started Have you go through a little bit through this slide? Back if you go back into the 1930s this in terms of the steroid hormones was the year of when we started just Isolating the chemical structure. So this was an era of the chemists Starting in the 30s at the same time What was going on in biochemistry in general and one could say in biology in general was the understanding that biology biological functions were due in particular to enzymatic machinery and these enzymes were protein in their chemical structure and What happened then was that in this year we started understanding glycolytic pathways oxidated pathways and so forth and Then we started realizing that some minor factors the vitamins particularly for instance your rival fluid But these are were important components of these biological systems in that they were actually part and acted as cofactors with certain other proteins and particularly a lot of enzymes so these Systems were being worked out then in the late 30s Now and also then by this time in the late 30s. We started realizing that some of the hormones were actually The metabolic products and in some cases metabolic products of other hormones so that testosterone was Found to be converted to estrogenic hormones, but just in a very general way Now then when we got into the 40s, of course, that was a time of the Second World War and Summer research was certainly slowed down during that period But again these various cofactors coenzymes these were kind of pretty much completely elucidated and People started developing sort of broader theories on this and David Green Who was actually on the faculty at the University of Wisconsin made this statement about any of these Trace element types of products like hormones and vitamins that they would function through their participating participation in enzyme systems now again over here we started seeing some of Evidence that when you injected a steroid hormones or looked at steroid hormone effects that you can see changes in Enzymatic activity, but it wasn't clear how the steroid was exactly doing this But it was just that there was a change in enzymatic activity following the administration of the steroid hormone so then we moved on to the 1950s and the 50s then were time of great revolution in biology and We've kind of finished working out some of the Mechanisms of Enzymatic function of metabolism and so oxidative phosphorolation electron transport were worked out the machinery for Protein synthesis was starting to be worked out and we started by the use of electron microscopy to see structures that have particular functions in these cells and This the climax of this sort of sequence of events in the 50s that was the Development of the double or development of the ideas of the double helix of DNA So that pretty much the structural machinery For how living systems work had been worked out by this time in the 50s Now in the case of the steroid hormones We now we're had available some radioactive we labeled the compounds and was shown for sure that the pathway then led from testosterone to estradiol and that one could look then at the estradiol going into tissues Plasma and coming through with the urine and feces and a large number of studies were being done on this Now remember that the C4 team labeled compounds in general are what the very low in specific activity And so these studies While useful in some ways We're not really leading to an understanding of how the steroid marbles function starting in the 50s particularly with bacterial systems the Steps from DNA gene expression through the intermediate of the RNA messengers and into the production of specific proteins was worked out and Things like the opera on theory and bacterial systems Alistair proteins coming out Beautiful work Shambos lab Not part of me shambos Stopped this I think to my questions No, I wasn't shambos So let me start up, and I'll just be starting this the same alabaster proteins were developed And then it was found that in animal cells that we had But appeared to be the precursors for messenger RNA But it was referred to as h n or hemogenius RNA because of the wide variety of sizes and so forth and at the time that was what all we could Do in terms of experimental work and then finally in 1965 a specific messenger RNA for hemoglobin was isolated Now what was going on with receptors in the late 50s? Estrogen binding was shown to occur in the uterus and other target tissues, and this was a very major Development and we'll spend some time talking about that and then in 1964 the nuclear localization of the bound estrogen was worked out and then 1966 there was the isolation of a specific cytosolic Protein that appeared to be the estrogen receptor and the development then of a model of which estrogen plus the receptor formed a complex and ended up being found in the nuclear compartment of target cells and Then in terms of tissue response again a lot of developments Estrogen effects were shown in general on RNA and protein synthesis And then it was shown that the effects of estrogen and a variety of things could be blocked if you block protein synthesis through the use of pharmacological agents in this case pure mycens and then similar studies were done with an RNA synthesis Inheritor actinomycin D. So the dependence on protein synthesis and RNA synthesis were shown again in this time frame and Then studies were shown that estrogen actually stimulated our memories activity and then this induction of specific Proteins were shown again by the middle of the sixties So in this period from the late fifties through the middle of the sixties a lot of developments Which laid the ground were for new ideas in this field occurred so this is sort of a synopsis of the of the Work I'd like to just follow up with now in this next slide We're showing shows a model of a system Which is rather of interest Historically, although probably of no practical interest and that is in the fifties if you have Been interested in hormone action and that's the time when I got introduced into this field Everyone knew how the steroid hormones work and this involved in both astrogens and The other steroid hormones such as androgens blue crocodiles And that was a theory that had been developed by a number of investigators which involved the concept that many of the steroid hormones had a Reducible hydroxyl group and in some position on the molecule in the case of estradiol Sort of major estrogenic compound. This is at the 17th position and this hydroxyl could be then Oxidized to astrome, which is another And the idea that was that in this system one would transfer Hydrogens and electrons to NADP to give you any to the reduced form of NADPH Getting a strong and this internment could read be in a sense recycled back to the estradiol Through the reduction from NADH now. This is you remember from your biochemistry is a system found in the energy producing parts of cells and NADPH is often thought to be associated with growth responses and is used as a source of electrons and reducing equivalents in growth processes So what we had was a system out of transferring Part of you reducing equivalents from this one system to this other system With the key intermediate being this trans hydrogenase with the cofactor being the Estrogen molecule in this particular case now Let's really fit into what everybody was thinking if you go back in time and think of the fact that everyone was concerned with Intermediate metabolism here was a very interesting system Now a few people raised questions about this and actually one studies this Total picture of the data in this system you realize that it's impossible to really explain all of steroid hormone action but it wasn't till a Organic chemist at the University of Chicago Elwood Jensen came along that this theory was disproved and Elwood Jensen's Elwood Jensen was an organic chemistry that at the University of Chicago But he was working in a laboratory of Charles Huggins who was a Nobel Prize winner for the Work that had been done on estrogen effects on breast cancer and in this setting Jensen brought in some interesting ideas To this system, but he made mostly brought an ability to do certain kinds of experiments and that is previous Work that had been done with radioactive hormones Injecting into animals that use C14 level compounds which have low specific activity Jensen had the ability plus he had the access to the Argonne laboratories near the Chicago Where he could make tritiated labeled compounds like gas exchange procedures and he was able to make them a high specific activity Estradiol Which had hundreds of times more higher specific activity than previously used C14 labeled estrogens So that was one thing plus he also was able to In an era before the scintillation counters that are currently used or other procedures. He was able to make use of procedures for measuring these low specific I mean these high specific activity estrogens in With Procedures and equipment that just was not available to very many people although shortly thereafter it was So he did just a very basic simple experiment. He just injected rats with the Label estrogen, but remember in this case he was injecting point zero one microgram Where most previous experiments have been probably working with Maybe close to a hundred micrograms of C14 act that active And What he found was that when he injected this material then took out tissues at various times afterward Their tissues fell into two categories tissues which Normally are thought to be target tissues for estrogen the uterus vagina that the There was an uptake period and then a retention indicating a slow decay or slow loss of the Estrogen from the tissue From these tissues whereas other tissues such as the liver kidney, etc There was a peak very early and then it declined very rapidly This was the first time anyone had seen any Data which suggested there was something unique about target tissues relative to non-target tissues in the way They interacted with the hormone or with any actual Tissue regulator so this graph here is probably one of the most key Pictures you'll see and that's from the work with Jensen did with a colleague in this case her Jacobson And this work was reported in the 50s and the interesting one interesting story that comes about this is That remember this was in an era where the transhydrogenases were were the answer to the problem And Jensen had this work done and he decided to give a Report at a meeting similar to the kind of meeting people are having here today and but in this case the meeting was an international biochemistry meeting in Europe and It was at the same meeting it turned out Fortuitously one might say or maybe not fortuitously By chance that there was a whole symposium on transhydrogenases and the spirit-harmonious Interactions with the transhydrogenases and their effects on it and this was a major symposium Probably a thousand people or so were at that meeting and again by chance Jensen's little ten minute talk was scheduled for the same time and so Jensen reports that a Handful of his friends. I've seen different numbers when it ranges from six to ten People were there in the audience to hear his talk and it's an interesting phenomenon to think about the fact that a major Symposium was occurring a lot of people went away probably thinking they had heard the gospel and There were ten people who had heard the data That pretty much Destroyed that whole theory rather quickly and there is a moral to this thing is that people then started Looking back at the transhydrogenase idea and started seeing it now emphasizing all the negative Things that happen for instance diethyl is still best of which is a very potent estrogen does not have a Hydroxyl group that can be oxidized and reduced by the same by any of these kind of enzymes that requires some different mechanisms Because it's a phenolic. They're all phenol phenolics hydroxyls and not The aliphatic type of hydroxyl So this Worked and really changed thing and then another critical thing that Jensen did is he took the extracts of these tissue with its radioactive activity that was present in the target tissue just ran it out on chromatography and just as you can see only seven different fractions and Either at two hours or six hours after injecting estradiol what he found was that 96 to 93 percent of the radioactivity Was still in the form of estradiol Tiny amount would be converted to estrogen so From this data he also then destroyed another old concept that had been present that is that metabolism of the hormones Was not crucial to their function This was a very major finding because at that time look at the bulk of the Work that was being done on the estrogens or any other steroid hormones In terms of their interaction with tire tissues involved looking for metabolites because of the again this Hero that we were in where metabolism was so important And this work they showed that it couldn't actually just this work alone Was not sufficient and Jensen did some other beautiful experiments and very cute experience. I've always thought which Again eliminated the possibility of there being any metabolism occurring in the estradiol in that target tissue Well then we worked out in this case starting off with work done by Bill Noteboom We started looking at Subcellular distribution of this radioactive material and It was quickly shown then that when you use physiological amounts of the hormone injecting that the Compound was present in nuclear fractions. This again was a revelation in that Again because again of some of the previous work on metabolism and so forth It was had been probably thought that in any cases of these hormones with function Perhaps not in the site of plasma compartment. Although Some people that already suggested that nuclear function is going to be important because of the work that our name protein synthesis But in any case this did prove that it was a nuclear function And I should say is that by the early 60s methodologies were available to measure steroid Trinium-labeled compounds and the trinium-labeled compounds have been made available by Some of the companies that were Producing these materials so now anybody could work with this Jensen's Work was not exclusive anymore. So this was then the nuclear function the next slide just shows immuno Are using immunological techniques With an antibody to the estrogen receptor that again in this case These are early embryos that the receptor is localized in the nucleus of these cells And this was in the nucleus whether there was estrogen or whether the embryos have been exposed to estrogen or not and in the case of the use of the Compot which would interact with it with this antibody rather specifically you can see how you Decrease the binding of the antibody and indicating some specificity to this Now the next step then was it turned out a little was present in the nuclear compartment after treatment if you took a tissue like the uterus and You ground it up extracted with a variety of media most of the receptor turned out to be Reveally extractable and was present in what we've referred to as cytosol fraction This does not mean that it was present in the cytoplasm. It just means that it was extracted from wherever I've been in the tissue What I and so people could extract the receptor a whole variety of techniques were tried to try to work with this compound Because of the peculiar nature of the receptor it turned out to be very very difficult and but David Toft Who's that now at the male foundation, but then was that at the University of Illinois? Was able to use sucrose gradients to separate out a fraction Which in this case Runs as you can see here as a very large protein or a protein aggregate and so here you would get this peak which was the Esther label estrogen bond to this protein fraction and with some binding then to An area where there was a bulk of the protein And here you can see the optical density then as an indicator this was where the bulk of the protein was present if you put in an Unlabeled estrogen in this case diethylstomestero, but also would be a s would I he would block the binding to this very heavy fraction and the only binding that occurred back here in this other fraction with this Ability to look at the receptor that in Cytosol and Finding most of it present in the nucleus there was another whole area of work that was going on And that was the response to the hormone now We said that there had been shown by the use of inhibitors like pure mice and an actinomycin D But there was a dependency on our own protein synthesis for the biological responses of the tissue And so there was an emphasis that I'm trying to find some more direct evidence for gene expression being involved and a variety of different Approaches were used it was we was able to show that there were effects of estrogen and in general an RNA Synthesis on protein synthesis this tissue is growing response estrogen. These are not surprising but Did not see anything rather specific, but Angela notates again at the University of Illinois did show in some very beautiful experiments That there was a specific at least one specific protein that would be turned down early. So in 1965 again back in Dark ages of Technology he used early gel electrophoresis and he would take tissue extracts from control and estrogen treated targeting tissues incubate one Say the control tissue with one isotope a C14 Isotope and then they on the other side he would then use a tritium labeled compound in With With the estrogen treated in this case He was using diethylstobesterole with tritium labeled proteins Use diethylstobesterole to stimulate the chip tissue and then incubated with a tritium labeled amino acid prekers And then a control over here In this case he's I should explain that The double labeling experiments were used in some situations in this particular case. He used a control System which we had two gels So we had tritium labeled amino acids in both case in this case in some cases We would use a C14 label To do these experiments for one of these situations. Then you would run the gels Try to run them in parallel as best you can if you could use the two different Isotopes and you can run them in one gel And what we found was that there was this protein that managed to move off into an area where away from the bulk of the proteins and it was markedly stimulated within an hour Of diethylstobesterole treatment or could be estradiol treatment And this protein then was referred to as ip protein and Later it was shown to be a protein of Some importance in intermediate metabolism But it's never been shown to have any major regulatory function But it was interesting that it did prove that there was a specific protein that could be induced by harm And then this led to many other people doing other systems some of which were more readily Worked with then this uterine tissue that that we were working with and this particular experiment So this all led them to a model In which the receptor is still the critical factor With estrogen being able to move into the target tissues and Finds these proteins which have a high affinity to estrogen and I haven't dwelt Dwelled I should say on that But the affinity of the estrogen for the receptor is extremely high and this was a crucial factor And this was worked out in these early studies back in the 60s early 60s and As a result of this interaction there's some modification in the receptor And in turn that this receptor then Has an effect on gene expression And then we can get these new proteins. So this was sort of the state of things in the 60s We now have obviously much more Interesting models one would say in that more complex models in which there are interactions between this Receptor here and with other systems of regulation that even occur at the cell surface So it's a much more complicated picture in a way. It was more fun Back when we had these simple models We didn't have to worry about all those complexities that you people are working in these fields Have to be concerned with in the present But this sort of general model held in the 60s And the outline of it is probably not too different than what is present now except with these additional complexities We thought at that time and we are we At that time that there may have been some kind of a translocation process a receptor into the nucleus Being regulated by the steroid I think that has been disproved At least in the case of the ester here receptor that appears to be localized in the nucleus prior to the interaction with the steroid hormone And So there are maybe some differences between different steroid hormones in terms of this initial localization of the receptor There's so many really interesting questions and start coming up as one starts dealing with this model for one thing is Why is this nuclear receptor as shown by the immunological studies and other procedures Why is it so readily extractable? What is it interacting with and under this set of circumstances versus once it's Been interacting with the steroid hormone Why now is it has such a high affinity for for the Nuclear some nuclear components So I'd like to then just end with again some of more of these questions that have kind of come up and Some things that you may want to be thinking about one again One of the major factors is the number of receptors that are present in the cell And in a number of cell systems, this seems to be in this nature In this range of 20 to 50 thousand per cell That means it's not a protein of in present Mass amounts on the other hand, that's an awful lot of receptors present if you think about stimulating one specific gene We don't think that it does Regulate one specific gene in most instances because in general it turns on growth and other things, but still it seems Like a surprisingly high number For even to regulate say a couple of other genes it seems like an awful lot of receptors And almost all of those which are the interactive with estrogen once Or once they have interacted with estrogen are bound in some tight form to some of the materials present And one other factor that's of some interest and this was from one system And that is uh twootary cells which respond to estrogen In two ways and one of these is that they respond in the production of prolactin And another way they respond is in is in growth There are some other systems that are rather similar to this, but this is the model that was used in our laboratory But that is if you have a fairly high concentration of estrogen You can occupy most of these by these binding sites And under those conditions you'll get Maximum growth and maximum prolactin synthesis But you if you start going down in the concentration of the hormone You'll get a lower occupancy rate And so at this 10 of minus 11th molar you have about half saturation of these receptor sites And if you move on down to 10 of the minus 14th, which is a pretty low amount of estrogen You're only going to get maybe 30 30 or let's let's just say less than a hundred of these sites occupy Now under those conditions Here you get essentially little or no Effect of the estrogen on prolactin synthesis If you get up here to 10 of minus 12th where you get 3000 or roughly 10 of the cells Occupied you'll start getting roughly 10 of the maximum prolactin synthesis effects What's surprising is to look here at the growth response and that is Even when you're only at half that maximum saturation you get about a 100 percent change in growth You can get all the way down here to this very small number of occupied receptor sites And you're getting half maximal effects on growth And this has been seen in at least two systems in this system and then in In mammary cells Which are which have been cultured for some period of time seem to respond in similar ways Although they don't have a simple marker like this, but in terms of their growth response They respond extremely low levels of the hormone So again, these are questions. Maybe we don't need all of these receptors for some effects And maybe we do for others or are there different pools of receptors that are being involved? And in any case here are questions like this and many others And that We're leading it to This next generation of people working on this to solve these problems Now if there is a question or two from another colleague Dr. Ferdinand Murdoch who's in the pathology department here University of Wisconsin kindly agreed to sit through this talk and Maybe raise the question or two and i'm going to sit down So the time of the 60s and the 70s is where you really emphasize How much new data was being collected and new ideas being put forth for how estrogen is worked What do you think were really the key concepts that got introduced during that time? I think again The work always seems to move in waves. The waves are dictated Somewhat by concepts, but actually mostly by By methodology and in that period we started finding learning how to work with the nucleic acids I think that was a major finding But also working more with proteins The ability to start separating proteins Even in the crew the gel electrophoresis that I showed at least one slide And starting don't look at RNAs we started People in the field started looking at at least ribosomal RNA as distinguished on this so-called heterogene Pardon me heterogeneous RNA And so this technology started permitting This Real ability to start looking at This these ideas and gene expression which were have been developed a lot in bacterial systems with where they could manipulate The systems in a lot different way, and it didn't really require all the biochemistry but the biochemistry Came after we developed a methodology to start looking at And it changed the direction because all of a sudden you didn't have to do it to me here in the town There's not to that point. That's about all you could do And you also emphasize the importance of having the most regular label ligands that you could follow the protein Right that was also I should that would be also extremely important aspect Particularly the trinium In the case of the steric armor and stuff because you just could not have done Anything without without them, you know the the whole concept of a receptor protein Or a receptor substance of some kind have been around since the turn of the century for neurotransmitters But they're real turn of the 19th century And but there really hadn't been a demonstration of a physical entity until I believe With the estrogen receptor by you and taunt the isolation of a fraction you would characterize On those sucrose gradients, but even up to that point there was resistance to the idea of a receptor Right the initial work came from the pharmacologist who needed to have a the at least a theoretical entity That you could explain how different drugs interacted with with tissues and with organisms And so that as you said came out extremely early, but people were unable to ever identify these compounds And so there was some skeptics actually in that memory in case of the A steroid receptor is even actually the interaction with purity nucleotides A steroid where it was thought at least by one very prominent Biochemical androgenologist And harvard was pushing that idea So the idea of what a receptor might be was really quite big and It was of interest that in some of the early meetings that I went to where there were Neurologists who were interested in neural receptors That the model system that was in use at that time were systems such as acetylcholine esterase, which is Really a disin-enzyme involved in the metabolism of a neurotransmitter But Certainly would be pushing it in terms of thinking of that being a receptor But those interactions were Were being studied as a model system for four receptors So it was really the steroid receptors were the first ones really to be any kind of a biochemical Although remember these were pretty crude mentioned those sucrose radians They were pretty pretty crude, but they were the receptor wasn't Isolated and you could do certain things with it Separated But I think during this time the protein chemistry actually gave us a lot of information about the steroid receptors and what they could Could do and maybe you could comment on some of the protein biochemistry that was done that Gave us the functions Right Defined a little more. What are you thinking of other things? Not only ligand binding, but DNA binding and all specific domains I think all of that was elicited by the protein chemistry. What's that? Yeah, a lot of a lot of a lot of the things that we now know about sequence analysis, particularly DNAs really had us roots back in these initial protein chemistry to where we We knew that the steroid receptors were related to each other just in the nature of the kind of compounds they found and many of their characteristics in terms of separations Techniques that were available at the time and later Development of DNA analysis much of this was was confirmed, but I would say to me at least welcome the ideas That the receptors were compounds, which certainly had at least some parts of them that were quite hydrophobic Those that turned out to be true That they are proteins that interact with other proteins to a great extent They're fairly complex and have domain structure And we knew already that there had to be something that would explain binding the steroid binding possibly others It's kind of interesting that there was some evidence already that there was some kind of allosteric type of protein that we were dealing with because We could show that there There was an influence from one binding site to another DNA Binding the steroid binding, but also the reverse of that DNA binding would have an influence on the steroid binding The other topic I'd like to raise is when you look back at the literature in the 60s 70s and 80s and certainly there's a lot of Was limited in how far it could go That idea actually goes I don't know actually the earliest reference, but one of the earliest references was What was in the work of Dr. Gerald Mueller University of Wisconsin, he had a model that he presented in 1957 at the Laurentian Armour Conference And in that model he indicated the estrogens We're interacting with something which in turn we're interacting with the chromat Target tissues and with a rather early perspective would be a kind of a crude model, but maybe it can contain the essence of Probably where we are at the present time where it's still a very difficult area to work But at that time of work on specific At least narrowing this down This was then in the 60s There's a whole PhD thesis On estrogen effects on chromatin On the histone proteins, but it's never been published It's very difficult to come up with real conclusions from With the limited kind of So I guess that comes full circle to what you said In answer to my first question, which is that these waves of Of understanding are driven very much by what we have available Technically to us Yeah, that's a big thing I think one of the big things I hope everyone may take away from this meeting Is the fact that you have to be careful on what What influences there are from the from your environment And science is not democratic You don't vote it In theories and stuff So genesis or the receptors Wasn't maybe the most popular thing in the world at the time I'm sure there were a lot of people But that was that that's what has prevailed and that's what you have to always be thinking about science So you want to Always be very suspicious be very skeptical of this mind feeling about About current trends because often they They lead you into Leaving stuff that is is not a certainty And I'll thank you and I think this is a good time