Bio 2B03 Module 6 Lecture 3 Script PDF

BIO2B03 scripts Module 6, lecture 3 Script Notes Slide 1 Welcome back to Biology 2B03, Cell Biology. Today, we will wrap up our Module 6 on Cell signaling, with Lecture 3, G-protein coupled receptors. So let’s get...

BIO2B03 scripts Module 6, lecture 3 Script Notes Slide 1 Welcome back to Biology 2B03, Cell Biology. Today, we will wrap up our Module 6 on Cell signaling, with Lecture 3, G-protein coupled receptors. So let’s get started! Slide 2 G-protein coupled receptors (or GPCRs) form a large family of receptors. Arguably, there are GPCRs involved in every physiological process in humans. Interestingly, a large number of diseases are also attributed to GPCR-related disorders. Given this, GPCRs are actually the targets of most pharmaceuticals that have been developed. Today, we will explore GPCRs, by looking at key steps in one, model GPCR signal transduction pathway. We will also identify similarities and differences between GPCR pathways and the cytokine and RTK pathways that we explored in our previous lecture. Slide 3 All GPCRs share a common structure: specifically, they have seven transmembrane alpha helix domains that loop through the membrane of the cell to form their final functional receptor. As a result, This creates four extracellular segments, E1 through E4, which will fold in the extracellular space to form the signal-binding domain. It also creates four cytoplasmic segments, C1 through C4, and these will fold to form an internal domain that interacts with a trimeric G protein. Well-studied examples of GPCRs include receptors that initiate stress responses, light-activated rhodopsins in the eye, odorant receptors in the mammalian nose, hormone & neurotransmitter receptors, plant growth hormone receptors, and the recent discovery of a glucose-sensing GPCR system in the yeast Saccharomyces cerevisiae. Nearly 800 different GPCR genes (or roughly ≈4% of the entire protein- coding genome) have been predicted from human genome sequence analysis, and as I previously mentioned, many diseases are the result of a compromise in the function of GPCRs. Slide 4 Of the hundreds of GPCRs in humans, the example we will look at today, is the GPCR-mediated pathway used in the stress response in mammalian cells. To start, catecholamines are water-soluble signals that circulate in the bloodstream. You may already be familiar with the most abundant catecholamines. These are epinephrine (or adrenaline), norepinephrine(or noradrenaline) and dopamine. Release of the hormones epinephrine and norepinephrine from the adrenal medulla of the adrenal glands is part of the fight-or-flight response. The catecholamine receptor to these signals is a GPCR. A s a w h o l e , the intracellular signaling pathway involves activation of the receptor- associated trimeric G-protein and with that, the activation of an effector protein, adenylyl cyclase. This adenylyl cyclase then modulates the cytosolic concentration of a secondary messenger, cyclic AMP or cAMP. With that, there are many cellular effects of increased cAMP concentration. All of these effects include an impact on the release of stored energy that is required for the fight-or-flight response. The fast response requires the activation of an enzyme, while the slow response will activate transcription. Let’s explore this further by looking at the adrenergic receptors. Slide 5 The adrenergic receptors fall into two subclasses, the alpha-2 receptors, and the beta adrenergic receptors. Epinephrine can bind to each of these two types of receptors, but it will induce different responses. These responses are also cell-type specific. This allows for coordinated responses to occur in different tissues in response to the same signal. The beta adrenergic receptors are stimulatory. With regards to tissue- specific location and function, activated beta adrenergic receptors in the liver and adipose cells stimulate glycolysis and lipolysis which are important for fuel mobilization. At the same time, activated beta adrenergic receptors on heart muscle increase contraction, resulting in increased blood supply to the tissues throughout the body. Finally, activated beta adrenergic receptors on the smooth muscle cells in the intestine have the opposite effect. Specifically, these activated receptors lead to an increase in smooth muscle cell relaxation so that all energy can be focused on fueling locomotory muscles during the fight or flight response, and so that it is not channeled towards unnecessary uses such as digestion, during times of stress. The alpha 2 adrenergic receptors are generally inhibitory. In particular, the alpha 2 adrenergic receptors are found in the cells of the blood vessels in the skin, the kidney and in the smooth muscle cells of  the intestine. Collectively the response is to cause the arteries to constrict and reduce the supply of blood to the periphery. Overall, the coordinated response of all of these receptors will increase the supply of energy to cells of the body that need to respond to stress. Slide 6 Where do these catecholamines come from? Well, catecholamines are products of the adrenal glands. These are small endocrine glands that sit on top of our kidneys, and produce a variety of different hormones such as epinephrine (or adrenaline), and the steroids aldosterone and cortisol. The epinephrine or adrenaline molecule, is shown here. Norepinephrine has a similar structure, however it is secreted by nerve cells and acts as a neurotransmitter. With regards to epinephrine, as a molecule, it can bind to both the beta and the alpha 2 adrenergic receptors, but while it is the same signal, it will induce different responses depending on the receptor to which it is bound. Along with this, the stress response increases the supply of ATP to cells through the breakdown of energy stores: this is glycogen and triacylglycerol. This is accomplished respectively in the liver, where glycolysis releases glucose from glycogen, and in the adipose tissue, where through lipolysis, this leads to the breakdown of fatty acids. Slide 7 We see here a cartoon of the membrane-associated proteins in the GPCR signaling pathway. To start, the seven-transmembrane domain GPCR (shown here in green) is shown in the inactive state. This is a state where the GPCR is not associated with a lipid- anchored, trimeric G-protein cluster. The other key player in this signaling pathway is the effector protein. An important note to keep in mind, is that similar to other G-proteins we have described, the trimeric G- protein follows the same principles of other monomeric G-proteins that we have seen. In particular, there is an active and an inactive state that is dependent upon guanine nucleotide binding. So, when the G-protein is bound to GTP it is active, and when it is bound to GDP it is inactive. So how do we activate GPCRs? And what happens then? Slide 8 Well, a GPCR is activated through binding of an extracellular signal. In particular, with binding to its ligand, this induces a conformational change in the intracellular domain of the GPCR, that allows it to interact specifically, and with high affinity to the trimeric G protein. The interaction induces a change in conformation that causes the dissociation of GDP  from the G-protein cluster, and allows binding of GTP in the nucleotide binding pocket. A s a r e s u l t , the GTP-bound G protein is active. Slide 9 From there, the trimeric G-protein dissociates, releasing the G- alpha subunit. This activated subunit can move laterally in the cell membrane and interact with the effector enzyme. The effector enzyme is shown in the activated state when it is interacting with the GTP-bound G-alpha protein subunit. This activated effector will remain active for a short period of time. That is only while the G- protein is associated. O v e r a l l , the length of time of the activation is dependent upon the intrinsic GTPase activity of the G- protein. F r o m t h e r e , once GTP is hydrolyzed to GDP, the G-protein becomes inactive. This in turn releases the G-alpha subunit from the effector, and inactivates the effector enzyme itself. The G-alpha subunit will then reassociate with the G beta and gamma subunits of the G protein cluster. Slide 10 As I mentioned, the Beta-adrenergic receptors are considered to be stimulatory receptors that stimulate cells to increase energy production, and utilization. Adding in some additional details here, these Beta-adrenergic receptors are associated with the stimulatory G protein, Gs. The Gs has three subunits, alpha, beta and gamma subunits. Just like all G-proteins, Gs cycles between the active GTP bound form and the inactive GDP bound form. In particular, upon receptor activation, the GTP- associated Gs alpha- subunit, dissociates from the trimeric complex and binds to and activates adenylyl cyclase. Adenylyl cyclase is the actual effector enzyme. A s a n enzyme, its role is to increase the intracellular concentration of the secondary messenger, cyclic AMP. From there, hydrolysis of GTP to GDP inactivates Gs alpha and it dissociates from adenylyl cyclase. In the absence of G-protein binding, adenylyl cyclase is then inactive. W i t h t h a t , in the absence of an active adenylyl cyclase there are ubiquitous or prevalent cytosolic enzymes that decrease the concentration of cyclic AMP. A s a r e s u l t , only by maintaining an active adenylyl cyclase through GPCR receptor activation can the cytosolic concentration of cAMP stay high. Slide 11 Adding in a bit more detail here as well, as I mentioned, the alpha2-adrenergic receptors are inhibitory receptors that inhibit the production of energy and energy usage. While these receptors have the same beta and gamma subunits as the beta- adrenergic GPRCS, these receptors are actually associated with an inhibitory G alpha, or Gi alpha, G- protein subunit instead. In the end, the Gi alpha  subunit interacts with a different region of the adenylyl cyclase catalytic domain, and inhibits its function. With this, in the absence of adenylyl cyclase function, there is no increase in cellular cyclic AMP levels. Slide 12 Summarized here are the two receptors with some sample stimulatory and inhibitory signals. Here, we see the beta adrenergic receptor on the left, and the alpha 2 adrenergic receptor on the right. As you can see, the beta adrenergic receptor will activate the Gs alpha protein subunit, which will stimulate adenylyl cyclase and result in an increase in cyclic AMP. In contrast, the alpha 2 adrenergic receptor will activate the Gi alpha -G protein, but activation of the Gi alpha protein will lead to an inhibition of adenylyl cyclase. We see here that different signals can activate each of these receptors. Because of this, this allows for the flexible regulation of cyclic AMP levels in a single cell. I n t e r e s t i n g l y , a s I m e n t i o n e d p r e v i o u s l y , epinephrine can activate both excitatory and inhibitory GPCRs on different cell types in order to coordinate diverse responses as part of the fight-or-flight response. Slide 13 What about the adenylyl cyclase enzyme itself? How is it making cyclic AMP? Well it does so, by converting ATP into cyclic AMP, while releasing diphosphate in the process. With that, as long as there is active adenylyl cyclase, the cell will maintain a high concentration of cAMP. Slide 14 But- a t the same time there is a constitutively active phosphodiesterase in the cell that counteracts the actions of adenylyl cyclase. T h i s m a k e s c y c l i c A M P u n s t a b l e i n t h e c e l l. This is because, phosphodiesterase catalyzes the breakdown of cyclic AMP into the non-cyclic form of 5’AMP. Interestingly, even as active adenylyl cyclase is making more cAMP, phosphodiesterase can also be breaking down cAMP. B u t , as long as adenylyl cyclase remains active, cytosolic cAMP remains high. However, as soon as adenylyl cyclase is inhibited, there is a drop in cAMP concentration due to the phosphodiesterase activity. Slide 15 Why does this matter? What does cyclic AMP or cAMP do? Well, cAMP is a small soluble molecule that plays the very important role of secondary messenger in cells. The concentration of secondary messengers fluctuates in the cell depending upon the activity of signaling pathways. In turn, it is the concentration of the messenger that determines that activation or inactivation of the next step in the signaling pathway. cAMP is an example of secondary messenger that responds to GPCR signaling pathways. W i t h t h a t , the concentration of cAMP modulates the activity of target proteins. This includes the activity of enzymes  such as Protein Kinase A or PKA. PKA is a serine/threonine kinase that phosphorylates a variety of target proteins. So how does cAMP activate PKA, and why is PKA important? Slide 16 Inactive PKA exists in a tetrameric form. There are two regulatory subunits shown here in green, and two catalytic subunits shown here in orange. On the regulatory subunits are nucleotide binding sites that bind cAMP. When cytosolic cAMP concentrations are low, there is no cAMP in these binding pockets. In this state, PKA is kept inactivate due to the interaction of the pseudo-substrate domain of the regulatory subunit with the substrate binding domain of the catalytic subunit. B u t - a s the concentration cAMP increases, the binding sites on the regulatory subunit are filled. This induces a conformational change in the pseudo-substrate domain of the regulatory subunit that releases the catalytic subunit. With that, PKA will now be available in an active form. Slide 17 At the top is a ribbon diagram that shows the interaction of the catalytic PKA enzyme with the pseudo-substrate of the regulatory subunit. Here, the pseudo-substrate region is shown in red. At the bottom is an illustration of how the pseudo- substrate domain changes conformation in response to cAMP binding and release. Again, the pseudo- substrate is shown in red. On the left, cAMP is bound and the pseudo-substrate retracts, allowing activation of the PKA enzyme (or the catalytic subunit). On the right, cAMP is released and the pseudo-substrate domain is extended and is able to block the substrate binding domain of PKA, thus inactivating enzyme function. Slide 18 But what are the targets of protein kinase A, and how does PKA regulation relate to the stress response? Ultimately the response to the epinephrine signal is an increase in the supply of energy to many tissues in the body. The role of PKA is determined by the targets of PKA activity. In particular, in order to release ATP, the body needs to supply glucose to the cells. R e m e m b e r b a c k to cell respiration that you learned i n p r e v i o u s c o u r s e s , t h a t the products of glycolysis (or glucose metabolism) are pyruvate and NADH. These are used by the mitochondria to make ATP. With that, remember that glycogen is a polymer of glucose and is a major source of stored glucose. Glycogen is synthesized by one set of enzymes, including enzyme glycogen synthase. This glycogen can then be metabolized or broken down into the glucose by another set of enzymes, including glycogen phosphorylase. During a stress response then, what we need to do in order to release glucose from glycogen is to inhibit glycogen synthase and promote glycogen phosphorylase. Slide 19 This is exactly what occurs. In the muscle, glycogen is metabolized into glucose-6-phosophate. This is the source of glucose for the cell. Glycolysis produces pyruvate and NADH, substrates for ATP production in the mitochondria. The increased supplies of ATP can power the skeletal muscles and cardiac muscles in the fight-or-flight response. In the liver, stored glycogen is metabolized to glucose- 6-phosphate. This occurs in by the indirect activation of glycogen phosphorylase, which catalyzes glycogen breakdown. PKA regulates this process by phosphorylating phosphorylase kinase, which in turn activates glycogen phosphorylase. In addition, the liver cells inhibit the production of more glycogen. This is accomplished when PKA phosphorylates and inactivates glycogen synthase, The liver cells can then release free glucose into the blood stream for transport to cells throughout the body. This is a fast, short-term response to epinephrine that requires only the modification of enzymes that are already present in the cell. Slide 20 There is also a slow, long-term response to this signaling pathway. PKA is shown here in its activated state. As you can see, the catalytic PKA subunit can also be translocated into the nucleus where it can phosphorylate transcription factors including a transcription factor called CREB. CREB binds to the cAMP response element or CRE. This is an enhancer sequence found upstream of many genes. When CRE is bound by CREB it enables the assembly of the transcriptional machinery to initiate transcription. W h i l e t h i s i s a s l o w e r , l o n g - t e r m r e s p o n s e , i n t h e e n d , the target genes include those that are required for the production of glucose, including the genes for phosphorylase kinase and glycogen phosphorylase. Slide 21 So in Module 6, we have looked at 3 examples of cell signaling pathways. Interestingly, while signaling pathways can be different, yet still share common features, the process of signal amplification is an important feature of all signaling pathways. This is because the signal can be amplified between more and more numbers of proteins or molecules that are present within a cell signaling pathway, signal transduction cascade. For example, with the stress response, between the addition of the epinephrine signal and the production of glucose, there is 108 fold amplification in response. T o s t a r t , the concentration of epinephrine is about 1/1010 molar in the blood system, but this is enough of a signal to activate a large-scale response in cells at many locations across the body. Examples of amplification  are seen primarily at steps that involve the activation of enzymes. For example, one adenylyl cyclase enzyme may produce 100 cAMP molecules while it is active, resulting in 100X amplification of signaling. Slide 22 And that’s all for Module 6. Today, we have seen that GPCRs are signal receptors that that span the membrane seven times. I n p a r t i c u l a r , they are associated with trimeric G-proteins that activate effector molecules in the cell. A s w e h a v e s e e n t o d a y , the effector molecule, adenylyl cyclase, moderates the cytosolic concentration of a secondary messenger called cAMP, and this second messenger in turn modulates the activity of enzymes such as PKA. I n t h e e n d , it is the targets of PKA phosphorylation that induce changes in cell behaviour including changes in metabolic activity and gene expression. Finally, we have seen a great example of GPCR function, by looking at how GPCRs can initiate the fight- or-flight response. In particular, GPCRs are able to mediate this response, by coordinating the release of stored glucose, for ATP production and use in our tissues.