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Lecture 11

Lecture #11

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University of Toronto St. George
Darrel Desveaux

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 thats 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 theyre 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. Its 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. - Were 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 were 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 wil
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