hey here we go a couple of things sorry I forgot to bring candy but it'll be on sale next week so we can probably bring it next week but I walking over I thought boy those of you who are here deserves some candy but I've just tried to sprinkle in a few interesting slides for your benefit I saw this on the MIT news of the day and I thought that was really cool who would have thought to turn the giant dome into a Halloween pumpkins and I was also jealous this morning when my husband got ready to go work in the emergency room and he put on his Star Trek outfit so I didn't even have it because I don't usually get to actually have a class on the day of Halloween or so he headed off I think people are unfortunately gonna expect him to be able to really fix things very readily in the emergency room today because he's gonna have all those extra powers that he doesn't usually have but anyway anyway so actually it's kind of a good day for a lecture Halloween because we're going to talk about the fight-or-flight response which is a great paradigm for cellular signaling so you're gonna see how signaling really works in action because what one has to think about with respect to cellular signaling is that it's dynamic and transient and when we look at the molecular details of the switches that enable dynamics and transient behavior in cells you're going to see how perfectly adapted they are for these types of responses that have to be carried out in cells are in organs in order to respond to a particular signal rapidly and with a definitive time frame and then have that signal then stopped once the the time frame is passed so I really want to sort of stress to you the characteristics of signaling that that can emerge just by knowing about two particular cell by cellular switches knowing the molecular details of those switches to understand then when we look at them in action in a couple of cellular signaling pathways then we'll see how adapted those signals are and the great thing about biology is the once you learn a few very specific things then those often get reused in nature so the cellular signals that I'm going to describe to you are used again and again in different formats to create different signaling pathways so it's not at every pathway in every cell in the body has different nuances it has general paradigms that we can learn about and understand all right so the key feature is then to think about the the the the molecular basis of these switches so last time I was talking to you about cellular signaling and remember there's always a signal a response and an output so something happens it's a signal it's usually a molecule of some kind outside the cell or able to diffuse into the cell as a function of that signal there's a response so this is molecular the response obviously is biochemical and the output is biological so I want you to sort of think about these as we look at pathways what's really my output at the end of a particular signaling pathway what was my input how did it get to the cell how does it have a dramatic effect on the cell as a whole how does the timing and action of this effect occur so rapidly so we talked last time about different types of signals those that mostly occur in this in the cytoplasm of the cell with signals that are able to diffuse across the plasma membrane and bind to an intracellular receptor and then cause an action but really the most important ones for today going to be the types of receptors that span the membrane and the reason why these are much more significant they're more in number they're more predominant is if you have a membrane spanning receptor you have the opportunity to use signals of very very different types small polar molecules small proteins lipids amino acids carbohydrates you've got a much larger range of signals than you could possibly have if you restricted yourselves to the type of signals that can cross the cell membrane those are very limited to nonpolar small molecules that can get across the membrane the more dominant types of signals are going to be the ones that are outside the cell they arrive at a cell surface they bind to attract a receptor that is trans membrane and transduce a signal from the outside to the inside so that's that's an important term here the process of transducing external information - internal information so when we started the course we were really thinking about laying down the cellular membrane as an encased environment where things could occur at high concentrations you could set up systems that were functional within membranes but in doing that within a membrane encased area in doing that you've built a formidable barrier around the cell so the types of receptors that we'll talk about are those that have adapted to take this external information into the cell and then have a cellular consequence occur so I'm going to talk to you about two specific types of cellular switches and they're going to be intracellular and we're going to be referring back to these because they're going to be important as we dissect a signaling pathway so the first type so stand aside a moment put aside a moment the actual process of a signal binding the response both biologic biochemical and biological let's look first at the molecular detail of these switches and see how they are adapted to their function and the first type of cellular switch i want to known as g proteins the G is because they bind guanidine nucleotides so that's why they're called G proteins they're small proteins that bind G DP or g GP so this is the guanine nucleotide that has either two phosphates guanosine diphosphate or triphosphate so there may or may not be a third phosphate here and the g proteins bind them and the dynamics of the situation are that when g proteins are bound to gdp they are inactive the switch is off there's an aspect of the structure and it's very dependent on how many phosphates there are in this structure but it's when it's bound to the diphosphate variant of the nucleotide then it's an off switch and when it's bound to gtp it's an on switch and it's active so this is a the molecular basis of one of the switches they relies on the the conformational dynamics of these small G proteins and when that shape is quite different if it's bound to guanosine diphosphate or triphosphate and we'll take a look in a moment and how the structure the shape changes the shape shifts upon binding the triphosphate analog so this is a dynamic inter conversion when the GTP is hydrolyzed you go back to the gdp bound state the off state and there are a variety of proteins that actually help these processes which we won't talk about in any detail the main thing that you want to remember is that when the g proteins are bound to gtp they're in a non state GDP they're in an off state and that's shown in this cartoon and here I should be able to if all goes according to plan show you the structure of a GTP analogue bound to a g protein so let's this little guys twirling around he's settled down a little bit the key thing you want to look at is where it's magenta and cyan the structure of the GDP and gtp bound g protein are very very similar but big changes happen in the yellow which is the gdp-bound form and the red which is the GT bound form let me go back again so you can see that one more time so in the GT gtp-bound form a portion of the protein swings around and binds to that third phosphate on the gtp and forms a different shape to the structure and that's a dynamic change that's responsible for activation when it's just D GDP that's shorter there's nothing for that red arm to bind and so it's much more of a floppy structure what I want you to notice in that yellow little yellow portion in the gdp-bound form you actually don't really see where the rest of the protein is this is because this is a crystal structure and in the crystal structure when things are very mobile you can't even see electron density it's as if the part of the protein isn't there because it is so dynamic it's only in the gtp-bound form it forms this tight compact structure that represents an on a switch that has been turned on does that make sense to everybody is everyone good with that so just that change that extra phosphate reaching further to the protein and making an interaction with the protein itself makes the difference in the dynamics of the g-protein and the activity here now there are different types of G proteins and you'll see both types reflected in this lecture there are small G proteins and they are monomeric and then there are slightly more complicated G proteins that are trimeric they have a hetero trimeric structure so they have quaternary structure where you have three different proteins as part of the complex so the other ones are trimeric and it's the G protein actually comprises three subunits where one of them is the important one that binds GDP or gtp but they're a little bit more complicated and in the first example when I talk about a particular response to adrenaline we're going to see the trimeric g-proteins and because they're trimeric that means there's three subunits and the convention is that they get the Greek lettering system so they are the alpha beta and gamma subunits they've each got their name they're three independent polypeptide chains and is actually the alpha subunit the binds GDP or gtp so that's one the formulation of one of the types of switches that we're gonna see when we start to look at a pathway what you need to remember here you need to focus on the fact that in one state the protein is in an off state it doesn't kick off a signaling pathway but in the other state the protein is a different shape because of binding a link let's just make this a little longer to that closed faith that's negatively charged to the protein so that's a non state and they're very definitive types of structures now both of these proteins are intracellular which means they're part of the response once a signal reaches a cell they're the things that change and they're what the the signal that gets transduced to the g proteins now there's another type of intracellular switch which is used very very frequently in nature and in fact it crosses permeates through all kinds of cellular processes and these is phosphorylation so here are the G proteins which is one and the other one is phosphorylation okay now remember we talked about reactions of proteins that alter their behavior their properties their dynamics so protein phosphorylation remember is a post translational modification a PTM it's something that happens to a protein after it has been translated and folded okay and the PTMs that a phosphorylation are involved OUP's amino acids that have OAH groups so that's the structure of tyrosine where the squiggles represent the rest of the protein and on phosphorylation we append a phosphate group oops - - to the oxygen on tyrosine on the sidechain so actually it looks pretty different it behaves pretty differently there are two other residues in eukaryotic cells that are commonly phosphorylated there the other two that include Oh H groups so they are serine and the third one to space but you get the general message and threonine so these are the three amino acids that commonly get a phosphate group attached and they change their properties they are called kinase kinase the root of the word is actually to change so the enzymes that catalyze this change are known as kinase a--'s and in contrast to the G proteins that use gtp the kinase is most commonly use ATP to give up a phosphate to phosphorylate the protein so there's another substrate in this reaction is ATP now when we look at this structure there's two or three things I really want to call your attention to once if we're dealing with this kind of switch we can go back to the off state by chopping up the gtp and making it GDP again so that's how to turn the light back off in the case of the kinase 'iz we've got to do something to go back from this state to the non modified state to turn the light switch off so for every transformation in the cell that involves a kinase there is a correspond reading set of enzymes that reverse the reaction called a phosphatase it takes that group off again so let me write that down here now phosphates used a lot so this is a possible protein phosphatase so kinase puts the phosphate on the phosphoryl protein phosphatase takes the phosphate off there are three types of amino acids that get most commonly modified in our cells tyrosine serine and threonine and one of the ones that forms an important part of an extracellular signaling mechanism are the tyrosine kinases and we'll delve into them in a little bit in the next second or but not the section I'm going to cover now but later because tyrosine various kinases come in a lot of different flavors the common flavors of whether you modify tyrosine or threonine serine because these are more similar to each other and this guy's different but we'll get to that later so in the cell we have about thirty twenty thousand genes that encode proteins including genes alright five hundred and fifteen of those are kindnesses that's a pretty big chunk of the genome you got to accept so in excess of 500 our kinase is specifically protein kinase is the ones that modify so there's a big chunk of the genome dedicated for this kind of activity and these dynamic because there's also about a hundred phosphatases they're a little bit more promiscuous you don't need so many of them but a large sort of component of the genome is responsible for phosphorylating proteins and d phosphorylating phospho proteins and that part of the genome it's own special name let's see I don't know and it is called Buckeye gnome I hope that's not too small in there so if we're describing all the enzymes in the genome that catalyze phosphorylation we would call it the kinase a collective set because it's the set of kinases and you'll hear that term quite quite commonly and the Tri no Ms really important and represents major major therapeutic targets because it's when kinase is go wrong that we have physiological defects so let's just go back to this you can see we've got the kinase and the phosphatase the donor for phosphorylation is ATP and it is the gamma phosphate that's transferred to the protein to switch it from the off state to the on state it is a post translational modification meaning it occurs on a protein after the protein has been fully translated there are a few Co translational PTMs that seems like a bit of a an oxymoron Co translational modifications but of phosphorylation ISM one of them glycosylation is and we won't go into those in any detail even though it breaks my heart not to go into those but ok alright so now I want to first of all introduce you to a paradigm for signaling as opposed to really go into what's happening and so one of the first paradigms is a situation where you have a cell in the plasma membrane of that cell is a receptor and it gets hit with a signal so a signaling paradigm is that a molecule from outside the cell binds to something that's transmembrane and then you start getting signal transduction through a pathway so any extracellular signal could be could be game in this process and then there's a sequence events upon signal binding there's a sequence of changes that ends you up with a final output and generally signaling pathways go through a number of steps where is there is the opportunity for the amplification of a signal so I talked to you last about time about some of the hallmarks of signalling specificity defines how how accurately that extracellular signal binds to the receptor but amplification really refers to how signals get bigger and bigger through certain steps in a signalling pathway in order to have a big impact in the cell not just a single event going through a single pathway one molecule at a time and we'll see that in the example that I show you okay then oftentimes when we look at signaling pathways we care a great deal about what's the first response upon the signal hitting the outside of the cell so in many signaling pathways this could be a protein the receptor could be a protein that is bound to a g-protein and that would be the first responder through the pathway that really triggers off the cellular events okay and now so what I want to talk about now is the the I always get this wrong I always thought it was flight or fight or fight or whatever right I always thought it was flight or fright but it's not the the response is actually the fight-or-flight so let's set this up to understand why this is such a great manifestation of a cellular signaling response because it includes a lot of the hallmarks that are really characteristic of the cellular response so this response involves a cellular receptor that is called a g-protein coupled receptor we saw a little bit about them last time they are always called GP CRS for short and what that term means is that it's a receptor that's linked in some way to a g-protein so it could be coupled to a monomeric or a trimeric g-protein so don't confuse the two one is the receptor it's transmembrane it's responsible for transducing receiving signals and transducing them the first responder is the g-protein that changes from a gdp-bound state to a gtp bound state and in the one that we're going to talk about we're going to deal with a trimeric g-protein in the fight or flight response and what you see here is a cartoon of the players that are involved so remember the G proteins we talked about them briefly last time they have seven transmembrane helix C's they span the membrane they have the n-terminal outside whoops one two three four five six seven and terminus out C terminus in and each of these is a transmembrane helix and this would be outside the cell this would be in the cytoplasm okay and you can actually often look at a transmembrane protein and know its behavior because the width of these transmembrane helix e's often comes in at approximately forty angstrom which is the span of a membrane you can sort of say that looks like a transmembrane helix it's exactly that dimension to cross a membrane and the trans the GPCRs in this case would bind to a ligand outside the cell and have a response inside the cell so those seven transmembrane helix E's are responding to ligand binding so let's take a look at this picture because it's almost impossible for me to get it onto the screen so that the its binds to a trimeric g-protein remember I talked to you about the two different types the trimeric has an alpha beta and gamma subunit quaternary structure and they're shown here in different colors the green is the alpha subunit the magenta is the or the red is the gamma subunit and the yellow is the beta subunit so what happens when the ligand binds to the g-protein there is a reorganization of that G Pro excuse me when it binds to the G protein-coupled receptor there is a reorganization of those seven transmembrane helix E's last time I identified them to you when you look at a couple of these they're actually fairly large loops that grab onto your ligand and that will translate conformational information through the membrane to the other side where the G proteins are sitting and in this response what happens is upon binding the ligand the alpha subunit leaves the team and goes from there GDP GDP bound States to the GT bound State so it literally changes its state and changes its mode of Association within the cell upon that action so you can see how nicely we have transduced the ligand binding out here to a pretty discrete cellular event turning on the switch of the g-protein alpha subunit is everyone following me there I know it so it looks complicated to start with B or C it in in action okay so here are the cellular components of the of the response so basically this is the kind of response where if you get scared or you feel you're in harm's way you will trigger this response in order to generate a lot of a lot of ATP in order that you can respond run away high to do something very active in order to rapidly respond to a threat of some kind and this response is triggered by a small molecule in this case it's epinephrine or adrenaline different names on different sides of the Atlantic but you all know what adrenaline is and here's the structure of epinephrine or adrenaline and it is the signal for the flight off right the flight or fight response because it's the small molecule that binds to the extracellular surface of the receptor changes its shape so that things can happen intracellularly and it's just one small molecule normally that would be charged it doesn't diffuse across the membrane so it's stuck being on the outside of the membrane okay so this is the signal that triggers the response so if you have to respond to a threat of some kind you can't stop sort of go to the fridge get a big snack eat it you know digest all your food and hope you're going to get energy quickly what you've got to do is have a response where you can generate energy from your glycogen stores that are in the liver so there is a signal comes from the adrenal region which is the release of adrenaline that goes to the cell surface receptors to trigger the response and what kind of signal would this be would it be paracrine autocrine EXO crying what kind of signal would that be oh sorry endocrine para crying Chuckster crime do you remember last time yeah endocrine so it's a response that comes from the kidneys and goes to the liver so it's going it's traveling autocrine is self paracrine is near juxtacrine is cell contact but any of these hormonal responses are pretty commonly endocrine responses so what happens once the signal binds so the specificity in this situation is that the g-protein coupled with now shown in pink in very stylized form but you can count those seven transmembrane domains will bind exclusively to this GPCR with high specificity another signal another small molecule that looks like it won't bind because we have to have specificity for the signal upon that binding event it will trigger a change within the cell and that change within the cell is that the alpha subunit here you see alpha subunit beta gamma they're all shown in green the alpha subunit of the g-protein remember I told you it was a trimeric g-protein where the alpha subunit is the key player the alpha subunit leaves the team and it exchanges its G GDP that's its resting state nothing's happening for gtp which turns it on so that's the first response the g-protein is responding to the signal from the outside of the cell through the auspices of the g-protein coupled receptor to give a change within the cell that's a discrete change okay following me so far so now what we need to do is trigger the remaining biochemical events that are going to get us out of this sticky situation where we need to produce a lot of ATP so it turns out that the gtp-bound form of the alpha subunit can then bind to another enzyme and that enzyme is adenylate cyclase so we've found we've changed the gdp to GTP there's a response and we activate the enzyme known as AC which you look up here it's called adenylate cyclase so this is a messenger within the cell that's now being generated as a response to the signal coming from outside through the GPCR to the alpha subunit of the g protein which then in its gtp-bound state binds adenylate cyclase okay is everyone with me and once that is bound adenylate cyclase can do its biochemistry and the biochemistry that adenylate cyclase as shown down here here's a teepee adenylate cyclase cyclize --is ATP you lose two of the phosphates and you get this molecule known as cyclic a MP which is a messenger molecule that will propagate information through the cell all right once that happens so now the adenylate cyclase is activated because it's bound to the gtp-bound form of the alpha subunit that means we can make a bunch of cyclic a MP and cyclic a MP is what's known as a second messenger and that often means it's a common messenger in a lot of pathways it shows up quite frequently within the wiring of a pathway and it acts locally to where the pathway is being is being processed so once cyclic a MP is formed from adenylate site by adenylate cyclase that then activates an enzyme it activates protein protein kinase a so PKA is a kinase it's actually a serine threonine kinase and that then results in certain proteins within the cell becoming phosphorylated to continue propagating our effect so we have specificity by the adrenaline binding we have amplification somewhere in this pathway so I told you that many pathways go through steps where you start amplifying the signal where do you think is the first stage in this set of transformations that I describe to you where you start amplifying the information think about what each of the events comprises what what is it what's you know is this one binding to one one event or is it one binding to one and we get multiple events where is the first step of amplification that's essential because it wouldn't do us any good if we make one molecule of ATP at the end of the day we've got to make dozens and dozens of molecules of ATP what what's the first event that could be an amplification over there yeah when it's made yes so this let's go through them one by pnes two one great once one binds to one one of these is released it gets converted to one of these once one of these is made adenylate cyclase is an enzyme so it can make a bunch of cyclic ANP which can then activate a bunch of protein kinase a which can then phosphorylate a bunch of cellular proteins so we got an expansion of our response all right so everyone does that make sense to everyone so amplification is really important feedback is also important if you ended up needing an EpiPen because you have an allergic response you might remember that you've got the jitters forever because there's too much firing an action going on but in though that the fight-or-flight response there's feedback at a certain stage that slows down this entire process and that feedback actually comes from an enzyme that chomps up the cyclic ANP to make it inactive as a second messenger so there's feedback in this process okay now what happens within the cell to get us that biological response this is a sort of a shortened version of what's happening so epinephrine binds here we are with the alpha subunit with cyclic A&P you know through each one of these you might make twenty molecules of cyclic ANP that would in activate many many PKS and then you go through a series of biochemical steps where different enzymes are activated with the overall goal of in the liver chewing up glycogen okay so glycogen is a pretty impenetrable polymer of carbohydrates and you need several enzymes to start to break glycogen down to make glucose phosphate and so these enzymes here of Prospero lays B kinase a glycation phosphorylase all end up converting glycogen into glucose 1-phosphate so you access your liver stores of stored carbohydrate which is in a polymeric form to get a lot of glucose phosphate which is then hydrolyzed to glucose which then hits the blood system and then you can deliver glucose to all the cells to go glycolysis and make ATP and every glucose molecule as you know can really turn out ATP so what we see in this process is going through the entire dynamics of the system where we've seen specificity amplification and feedback later on I'll describe integration to you ok everyone following the series of steps that go from a Maalik molecular messenger to biochemical steps to physiological biological response now I want to just emphasize one quick thing here I've got a couple of slides I popped in of drug targets about 45% of receptors in cells 25% of the entire drug targets are GPCRs they respond to all kinds of signals amines amino acids lipids little peptides proteins nucleosides all commonly going through the g-protein coupled receptor to give you a similar phenomenon to what I've described to you and what I thought was think is particularly interesting this is not I'm gonna post all of these are slides what I want yous to see this was quite a while back but it just shows you so many of trademark drugs that targets different gpcrs they're shown here and what diseases they're used for to treat and what's the generic name of those drugs so you can see here many many diseases have at the heart and soul of their the problem different receptors and these are all g-protein coupled receptors that are treated with small molecules the bind to the and often glue it in an inactive state so it can't then bind to an activating signal and have all the rest of the events occur there are very few structures of the g-protein coupled receptors but there are some of them so many of the target g-protein coupled receptors can be modeled computationally and then you can do a lot of work where you actually model the receptor in a membrane environment and start searching for drugs through computational approaches and I thought a lot of you might be interested in this because this is a really strong axis where bioinformatics confirmation and advanced physics in molecular dynamics can be brought to bear on drug discovery when you don't have perfect molecular models of your targets okay so now we're gonna move to a different kind of signal we're gonna talk about the receptor tyrosine kinase ISM alright so in the receptor tyrosine kinase responses we can often see very similar paradigms to what I've just shown you but there is an important distinction receptor tyrosine kinase is we often call these are T Ches so that's their shorthand so over here I described to you two different kinase 'iz that we have kinase a--'s that modify threonine serine and tyrosine the receptor tyrosine kinase is a subset of tyrosine kinase --is that form part of a receptor so if you were to think about various kinase 'iz you would have the serine threonine and you would have the tyrosine ones but these would be differentiated into the ones that are parked part of a membrane protein the rtks and then the ones that are soluble in the cytoplasm and the serine threonine ones are most commonly soluble in the cytoplasm I'm going to focus on the receptor tyrosine kinases because they do slightly different activities when they signal relative to the GPCRs so let's once again this situation another paradigm where you see a series of events but with a number of the receptor tyrosine kinase pathways the ultimate action ends up being in the nucleus where as a result of an extracellular signal you get a series of events that ends up with a protein being sent into the nucleus and that protein may be a transcription factor the binds to a promoter region and as a result of that you'll get gene transcription occur you'll transcribe a gene make a messenger RNA that will leave the nucleus and cause action within the cell so this is a little bit different than the other response that was mainly cytoplasmic okay so let's take a look at the receptor tyrosine kinases receptor tyrosine kinases are proteins that span the membrane but rather differently from the gpcrs and they have a domain that's extracellular just a single transmembrane domain this is out this is in and then they have an intracellular domain this would be where the ligand binds this would be how there's some kind of signal transducing and this would be a kinase domain okay so how do we get the information in when we saw the gpcrs we saw the ability of those 7tms to kind of reorganize and send information in the cell with the receptor tyrosine kinase --is it's kind of different there are regions of the membrane where there are a lot of these proteins they commonly bind small peptides and protein molecules and when they're in their activated form once the small protein binds the receptor tyrosine try forms a dimeric structure that is two of these get together only upon ligand-binding they move together once there is a ligand bound and then what happens is that the tyrosine kinase domains phosphorylate each other and that's activation in the case of receptor tyrosine kinases so when the small protein ligand is not around this is a singleton it doesn't work on itself once this ligand binds interactions change you get a dimeric structure where one kinase can cost correlate what's called in trans the other kinase domain so it's different from the GPCRs it's a different kind of feel to it but it's still a dynamic transient signal let's take a look at this within a cell and see what kinds of responses and this is in our response to EGF which is epidermal growth factor it's a cytokine that promotes cell division so a lot happens with respect to the action of a cell not to produce ATP but now to respond by producing all the elements that enable cells to grow and proliferate so the epidermal growth factor binds you get dimerization upon that dimerization the kinase domain in one structure in the blue one phosphorylates the other and vice versa they phosphorylate each other intermolecular ly once that has happened through the auspices of another protein i won't bother you with the name of this phosphorylated intracellular RT k binds to a small g-protein in this case it's a monomeric g-protein not one of the trimeric ones a small one known as wrasse once that binding event occurs guess what wrasse gets activated it's now binding GTP instead of gdp and then it starts going through a sequence of events where there's a ton of cellular phosphorylate controlled cellular phosphorylation events the result in moving a protein into the nucleus that helps form a transcription complex the results in cellular proliferation similar but different series of events they're still amplification they're still dynamics and in this case it's a lot of phosphorylation events and what I want to sort of define for you is that many of these pathways are in trouble in disease state speed inflammation neuro degeneration or cancer there are a Bearnaise aberrant behavior of proteins within these pathways that cause them to go wrong cause cells to proliferate out of control or undergo bad responses and that is why these proteins end up being therapeutic targets like the g-protein coupled receptors okay so we've seen the characteristics of signaling we've seen a signal we've seen amplification we've seen responses but I just want to quickly show you is an idea about integration so here's an idea with two signaling pathways that sort of end up with the same signal outside where you integrate actions through two different signaling pathways to achieve a bigger different kind of response so that's that last hallmark of signaling pathways it's not that every pathway is clean and straight it has crosstalk with other pathways and you get amplified or different responses tremendously complicated I want to give you one more term and then I'll show one table when these pathways go wrong it's often because switches get stuck on so for example a gtp a g-protein gets stuck in its gtp bound state or doesn't even need gtp to be activated or a tyrosine kinase is stuck activated and that's what's called constitutively active I mean base basically meaning it's permanently on so many of the diseases that are caused by mutations in your genome not genetic diseases but mutations in your genes in some particular cells end up with constitutive activation where you don't need a signal to have a response and so for example cells may proliferate out of control so that is an important term to know and understand because constitutive activation basically means that a receptor may be active in the absence of a ligand and I believe this is my last slide I just wanted to leave you with this when one thinks of GPC ours their tremendous therapeutic targets the world of kinase is is no less important these scale is in billions of dollars spent on developing molecules that may be curative of diseases that involve dysregulated signaling and what you see on this I want to point out two things I'm going to go in of course this thing stopped working at the last minute but what I want to point out is this particular bar this represents the billions of dollars spent on protein kinase inhibitors over-fire a five-year period and it's just escalating and escalating similarly monoclonal antibodies are very important but the small molecule drugs hold a real dominance what do these drugs do they enable you to have small molecules that can go into dysregulated signaling pathways and stop the activity somewhere in the pathway to avoid signals going constantly to the nucleus and turning things on all the time so that both of these types of functions in cellular signaling once you want to understand both from a biological perspective but from a medical perspective okay you