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Lecture 11

BIO241 Lecture 11

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Jennifer Harris

Tuesday, February 10, 2009 - We continue with cell communication and basically how cells communicate with each other. - Recall from the last lecture we went through an overview of different receptors found in animal and plant cells. We went through the example of G protein coupled or G protein linked receptor. - We will continue on with G protein linked receptors today. Recall that G protein linked or G protein coupled receptor is membrane associated usually with 7 transmembrane domains & it has a ligand binding domain at the N terminus which also has a component of the ligand binding domain usually that’s part of one of the transmembrane domains. - Now normally it is inactive in the absence of the ligand but when the ligand binds, there is a conformational change in the G protein coupled receptor that leads to activation or conformational change on the cytosolic side of the G protein coupled receptor thereby activating it. - Now these G protein coupled receptors are often associated with trimeric G proteins. Here is a blow-up of one of these trimeric G proteins, it is made up of 3 subunits: alpha, beta and gamma. The alpha and the gamma subunits are associated with the PM through fatty acid modifications so they’re anchored in the plasma membrane to these fatty acid modifications. - The alpha subunit is a GTPase. That is why these are called trimeric G proteins, these have GTPase activity or the alpha subunit has GTPase activity. In the inactive form, the alpha subunit is bound to GDP shown in the slide. - When there is an extracellular signal or ligand that binds to the G protein coupled receptor, it induces a conformational change, activates the receptor. This is transmitted to the cytosolic side and now the G coupled protein receptor can activate the G proteins. - The G proteins, the trimeric G protein in this case, the alpha subunit, the GDP is replaced for a GTP, this leads to activation of the trimeric G protein and then what happens next is these two components, the alpha subunit and the beta-gamma subunit are then activated and can go on and activate different cellular components. We saw how the alpha subunit can activate adenyl cyclase last time. - He knows there is discussion about whether the alpha and beta-gamma subunits stay together, in some cases they will stay together and they can activate components together, and in many cases, they separate from each other so the alpha subunit can go and activate a specific cellular target and the beta-gamma subunit can also go and activate a cellular target. It’s not that important whether they act together or not, it is important to know that they become activated by the G protein coupled receptor and both the alpha and beta-gamma subunits can then go and target their cellular targets. VIDEO - So we had tried to get this movie working last time and it goes over basically G protein signalling and it will work today. - Many G protein coupled receptors have a large extracellular ligand binding domain. When an appropriate protein ligand binds to this domain, the receptor undergoes a conformational change that is transmitted to its cytosolic regions which activate a trimeric GTP binding protein or G protein for short. - As the name implies a trimeric G protein is composed of 3 protein subunits called alpha, beta and gamma. Both the alpha and gamma subunits have covalently attached lipid tails that help anchor the G protein in the plasma membrane. In the absence of a signal, the alpha subunit has a GDP bound and the G protein is inactive. - In some cases, the inactive G protein is associated with the inactive receptor while in other cases, it only binds after the receptor is activated. In either case, an activated receptor induces a conformational change in the alpha subunit causing the GDP to dissociate. - GTP which is abundant in the cyotosol can now readily bind in place of the GDP. GTP binding causes a further conformational change in the G protein activating both the alpha subunit and the beta gamma complex. In some cases, as shown the activated alpha subunit dissociates from the activated beta-gamma complex whereas in other case, the two activated components stay together. In either case, both of the activated components can now regulate the activity of target proteins in the PM as shown here for a GTP bound alpha subunit. - The activated target proteins then relay the signal to other components in the signalling cascade. Eventually the alpha subunit hydrolyzes its bound GTP to GDP which inactivates the subunit. This step is often accelerated by the binding of another protein called a regulator of G protein signalling or RGS. - The inactivated GDP bound alpha subunit now reforms an inactive G protein with a beta-gamma complex turning off other downstream events. - As long as the signalling receptor remains stimulated, it can continue to activate G proteins. Upon prolonged stimulation however, the receptors inactivate even if their activating ligands remain bound. In this case, a receptor kinase phosphorylates the cytosolic portions of the activator receptor. Once a receptor has been phosphorylated in this way, it binds with high affinity to an arrestin protein which inactivates the receptor by preventing its interaction with G proteins. Arrestins also act as adaptor proteins and recruit the phosphorylated receptors to clathrin coated pits from where the receptors are endocytosed and afterwards, they can either be degraded in lysosomes or activate new signalling pathways. - So that goes over how G proteins coupled receptors transmit their signals to the G proteins & then they go on & activate target proteins & we looked at one of those target proteins & that is adenylyl cyclase.  Phosphorylate specific substrate proteins  Proteins phosphatases dephosphorylates target proteins - An adenylyl cyclase will produce cyclic AMP from ATP in the cell. - What happens downstream from that, how does cyclic AMP actually transmit a signal to downstream signalling components in the cell? - This is just an overview, here would be the activated G protein coupled receptor bound to its ligand, this will activate the G alpha and the beta gamma complex. These, especially the G alpha subunit when it is activated can go and activate an adenylyl cyclase molecule. Then the activated adenylyl cyclase will produce cyclic AMP from ATP. - What happens next in many cell types, one of the targets of cyclic AMP is a protein kinase called protein kinase A. Protein kinase A will be activated by the presence of cAMP in many cell types & then it goes on to do many different things depending on what the cell type is. - We’re just going to go over one example to give us a fundamental understanding of how protein kinase A is activated and then transmits the signal down to its downstream components. - Protein kinase A can basically be divided up into two components: two catalytic subunits and two regulatory subunits. In the absence of cAMP, the regulatory subunits are bound to the catalytic subunits and this forms an inactive protein kinase A molecule. Once cAMP gets into the cell, cAMP binds to the regulatory subunits of protein kinase A and then regulatory subunits will dissociate from the catalytic subunits. This is induced by the binding of cAMP to these regulatory subunits. Once the catalytic subunits are released, these are active and this is what is called activated protein kinase A. These catalytic subunits will then go to phosphorylate specific substrates or proteins in the cell and these will vary depending on the type of cell we’re dealing with. - There are two major levels of the activation that can occur by PKA. One of them is very fast activation of specific responses and this will basically be the phosphorylation of a specific kinase, for example or specific protein that then goes on and degrades glycogen for example or is involved in other metabolic processes. That is a very rapid response, PKA phosphorylates something and the target does something immediately to benefit the cell. - There are also slower responses, we will have examples of both of these. Slower responses require transcriptional changes in the cell so PKA needs to activate a specific transcription factor and then that a transcription factor will activate or transcribe specific genes in that cell type and then those transcripts will produce proteins that will produce the metabolic response of interest. Those are slower responses so two different levels of responses. - The effects of PKA on cells are transient. The reason for that is that once PKA phosphorylates its target proteins, there are many protein phosphatases in the cell that will dephosphorylates those proteins and inactivate them so PKA is phosphorylating those target proteins to do something, activate them or in some cases inactivate them, but then there are phosphatases that are antagonizing that effect by removing phosphate groups. The effects of protein kinase A are very transient due to the presence of these protein phosphatases that will remove the phosphate groups that have been added.  To promote glucose release  Promote breakdown of glycogen  Inhibit glycogen synthesis - One cell type we’ll go into more detail is liver and skeletal muscle cells where PKA plays an important role. - Now in these types of cells one of the major storage forms of glucose in animal cells is called glycogen. - Adrenaline or epinephrine, one of the responses to adrenaline is the release of glucose from these cells from the glycogen stores so how does adrenaline, which is the ligand for G-protein coupled receptors actually activate or induce the release of glucose from glycogen stores? So the response is to promote glucose release from these glycogen stores. - Here is the G protein coupled receptor, the signalling molecule in this case would be adrenaline, the activated G protein coupled receptor would activate the alpha and beta-gamma subunits, the alpha subunit can then activate adenylyl cyclase to produce cyclic AMP. The cAMP activates protein kinase A, the released catalytic subunits then go on to do two things to promote glycogen breakdown. - One of them is that PKA phosphorylates glycogen synthase, now glycogen synthase is actually the enzyme that is involved in synthesizing glycogen so by phosphorylating glycogen synthase, PKA actually inhibits the activity of glycogen synthase and therefore cannot synthesize glycogen so you inhibit the synthesis of glycogen which is the second point, inhibit glycogen synthesis through the phosphorylation of glycogen synthase. - The second effect is to promote the breakdown of glycogen so inhibit the synthesis and promote the breakdown. The way PKA does this is to phosphorylate this protein glycogen phosphorylase kinase that is also activated by phosphorylation and then this kinase will phosphorylate and activate glycogen phosphorylase and that is the enzyme that breaks down glycogen so glycogen phosphorylase will break down glycogen to glucose 1 phosphate. - Now glucose 1 phosphate will be converted to glucose 6 phosphate and that can be used in the glycolytic pathway to produce ATP for that cell. - This is a very rapid response, PKA is activated and then it phosphorylates two enzymes involved in glycogen metabolism and that releases glucose from glycogen by inhibiting glycogen synthesis and promoting glycogen breakdown. - A slower response induced by PKA occurs in the liver and basically the liver and other cells also. PKA will alter the transcription of numerous different cell types but in the liver there is the transcription of an enzyme that will actually convert glucose 6 phosphate into glucose, into free glucose that can then be released into the bloodstream. How does this occur?  Activate transcription of target genes - In certain cell types, activated protein kinase A normally found in the cyotosol can translocate into the nucleus and once inside the nucleus, the activated protein kinase A phosphorylates a transcription factor called CREB. In nonphosphorylated form, CREB is inactive. - Now CREB stands for cyclic AMP responsive element binding protein. Now in these cells, there are a number of genes that are activated by the presence of cyclic AMP and they have a promoter element called this CRE or cyclic AMP response element and basically this element allows genes to be transcribed in the presence of cAMP. Now this transcription factor CREB binds to these elements, these CRE elements and that is why they’re called CRE binding proteins. - CREB will be phosphorylated by activated PKA in the nucleus and then phosphorylated CREB will then recruit this additional protein called CREB binding protein or CBP and it is the recruitment of CBP that will activate the transcription of these target genes. The recruitment of CBP only occurs when CREB is phosphorylated. In cells where CREB is not phosphorylated CBP is not recruited. - Once CBP is recruited, there will be transcription of these target genes & one of these is glucose 6 phosphatase & this is the gene in liver cells that converts glucose 6 phosphate to glucose & then glucose can be released to the blood stream & be made available to other tissues like the brain & muscle. - This is a slower response to activation of PKA but both of these responses are the result of the action of cAMP on protein kinase A. - Okay, this is another animation that nicely shows the activation of PKA by cyclic AMP. - Adenylyl cyclase is a membrane enzyme whose catalytic domain is activated by the GTP bound form of the stimulatory G protein alpha subunit or G alpha S for short. - Activated adenylyl cyclase converts ATP to cyclic AMP which then acts as a second messenger that relays the signal from the G protein coupled receptor to other components in the cell. - In most animal cells, cyclic AMP activates cyclic AMP dependant protein kinase or PKA. In the inactive state, PKA consists of a complex of two catalytic subunits and two regulatory subunits. The binding of cyclic AMP to the regulatory subunits alters their conformation and liberates the catalytic subunits which are now active and phosphorylate specific target proteins. - In some endocrine cells for example, the activated PKA catalytic subunits enter the nucleus where they phosphorylate a transcription factor called CREB. Phosphorylated CREB then recruits a CREB binding protein. This complex activates transcription after binding to specific regulatory regions that are present in the promoters of appropriate target genes. - We’ve covered G proteins coupled receptors taking you from the outside of the cell, the signal, through the receptor, through the different cellular signalling proteins such as adenylyl cyclase and PKA down to the regulation of gene expression at the transcriptional level through the phosphorylation of CREB. As you can see, the nice transmission of the signal from the cell surface down to the nucleus. - That is G protein coupled receptors and now we move on to the second category of receptors called enzyme linked cell surface receptors. - If you’ll recall from last lecture, these enzyme linked receptors basically are also membrane spanning and they either have intrinsic enzyme activity or they’re associated with specific enzymes often these are kinases. So these receptors, the ones that we’ll be dealing with either have intrinsic kinase activity or are closely associated with kinases on the intracellular side of the membrane. - What usually happens is these receptors are found in the monomeric form when they’re not bound to their ligand. When the ligand comes in, this can be a growth factor, lots of these are growth factor receptors such as epidermal growth factor receptor that we saw previously in previous lectures. When the ligand binds to the receptor, it often induces dimerization of the receptor and the dimerization will activate the catalytic domain of these receptors. Or in the case where they’re associated with the kinases, the dimerization will activate the associated kinase. - Once this enzyme activity is induced, then it can go on and it will modify downstream components in the cellular signalling pathway so the first example we’ll look at are an important class of enzyme linked receptors or enzyme coupled receptors called receptor tyrosine kinases in animals.  RTK dimerization  Phosphorylation of kinase domain  Binding of intracellular signalling proteins - These receptor tyrosine kinases have tyrosine kinase activity so they have intrinsic to them, a tyrosine kinase domain and the ligand for these are growth factors and hormones. The response is often cell proliferation, differentiation and survival. For this reason mutations in these pathways can often have important roles in certain cancers. - The receptors can be divided into two major domains, an extracellular domain which is outside the cell shown and this is what binds the ligand. Then there is this cytosolic domain shown here inside the cell and this is what has the enzyme activity and in this case, the enzyme activity is a kinase domain which is a tyrosine kinase domain meaning that it will phosphorylate tyrosine residues on proteins. - Once the ligand binds, this could be growth hormone for example and what it will do is it will lead to receptor tyrosi
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