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Biochem 285 FA23 E. Mearls Class Activity #9 Total Points = 24 Your Name:Devanshee Sanghvi I worked on this assignment independently ☐ I worked on this assignment with: Keerti, Joyce ☐ Part I: GPCRs (8pts) You are studying cell signaling through GPCRs. Specifically, you have identified two previously uncharacterized GPCRs called GPCR-A and GPCR-B. A) You have found that activation of GPCR-A activates adenylyl cyclase, but activation of GPCR-B inhibits adenylyl cyclase. How is this effect being mediated in the signaling pathway? (2pts) This effect is mediated by signal molecules which bind to the receptor to activate the GPCR. Signaling molecules are specific to the receptor it can to which is associated with a type of GPCR in this case there are 2, A and B. B) GPCR-A and GPCR-B are occasionally, but not always, found in the same cell at the same time. Is it reasonable to assume that they could both bind the same signaling molecule? Explain your reasoning. (2pts) It is not reasonable to assume that they could both bind the same molecule. This is because the activities or the effects on the signaling pathways of the 2 GPCR are opposites. GPCR-A activates adenylyl cyclase which activates the pathway while CPCR-B inhibits adenylyl cyclase which inhibits the pathway. If they can bind to the same signaling molecule they would be turned on at the same time which would be a waste of energy as it has opposing effects and competes. C) What is the outcome each mutation below would have on the amount of cAMP? Note: “G-protein A” is the G-protein that works with GPCR-A and “G-protein B” is the G-protein that works with GPCR-B. You can assume that GPCR-A and GPCR-B are being activated by a signaling molecule in each case. (4pts) Modification Effect on [cAMP] (will cAMP increase or decrease relative to a non-mutated control) 1 G-protein A is unable to hydrolyze GTP cAMP will increase - always on which means activation of adenylyl cyclase → more production of cAMP. 1

2 G-protein B cannot release GDP cAMP will increase - cannot activate which means will not be able to inhibit adenylyl cyclase. 3 The RGS that targets G- protein B is non- functional cAMP will decrease - RGS is the regulator, stimulate the hydrolysis of GTP to turn protein off. In this case G- protein can not be turned off. 4 GPCR-A cannot interact with G-protein A cAMP will decrease - In order for the G-protein to become active it must interact with the receptor in order to release GDP and bind to GTP through a change of confirmation. Part II: Insulin vs. glucagon (7pt) A) Insulin and glucagon are peptide hormones that must bind to a cell surface receptor. Why can’t they simply diffuse through the plasma membrane and activate targets in the cell? (1pt) Glucagon and insulin are large polar molecules that cannot easily diffuse through the plasma membrane; therefore the hormones are repelled by the hydrophobic tails of the plasma membrane. As a result, they cannot easily diffuse across the membrane and require binding to a cell surface receptor. B) Insulin and glucagon have opposing effects on several key metabolic enzymes in cells. One of these enzymes is glycogen phosphorylase (GP), an enzyme that catalyzes a reaction in the pathway that breaks down glycogen to release glucose. Glucagon activates a kinase (PKA) that activates another kinase called phosphorylase kinase (PhK). Insulin activates a phosphatase (PP1). Both PhK and PP1 target GP and alter its phosphorylation state. Given this information, what can you infer about the activity of GP when it is phosphorylated vs. when it is not? Explain your reasoning (3pts). When GP is phosphorylated, it is activating because it is able to trigger a phosphorylation cascade. When glucagon activates PKA, activated PKA is allowed to phosphorylate target enzymes. When insulin activates phosphatase PP1 and dephosphorylates PKA, the protein is unable to phosphorylate target enzymes. 2

C) People with Type II diabetes are usually insulin resistant, which means that their cells do not respond very well to insulin even if it is present. As a result, their blood glucose levels are usually higher than a non-diabetic person. Use what you know about what occurs in the insulin signaling pathway to explain why type II diabetics usually have higher blood glucose levels (3pts). In the presence of insulin, glycolysis and glycogen synthesis is activated while gluconeogenesis and glycogen degradation is inhibited. Cells of Type II diabetes patients are unable to respond to the insulin which means it struggles to activate glycolysis and glycogen which is the catabolism and storing of glucose, respectively. Furthermore, it will have trouble inhibiting gluconeogenesis and glycogen degradation which is the creation of glucose from pyruvate and breakdown of glycogen to glucose. As a result, glucose is high in the cells which results in a high blood glucose level. Part III: Application – Cell signaling and Disease (9pts) You are a scientist studying two related congenital diseases, Lion syndrome and Tiger syndrome. These syndromes are characterized by developmental defects of the heart and skeletal system, as well as a greater risk of developing cancer. Some cases of Lion syndrome are caused by activating mutations in the protein, Raf. The genetic bases of Tiger syndrome are unknown. Your team working to identify drugs that can treat these diseases. Drugs that inhibit Raf have been shown to produce positive outcomes in cancer patients with Lion syndrome. 3

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You find that Tiger syndrome patients also respond well to Raf inhibitors, so you sequence the gene for Raf in several Tiger syndrome patients but find no mutations. Based on what you know about the MAPK/ERK pathway (shown above), you sequence the Sos gene in Tiger syndrome patients. You find that 5 out of 10 patients in your trial have a mutation in the Sos gene. Introducing this mutant version of Sos into cells in culture leads to increased levels of phosphorylated ERK compared to controls. a) Given these results, how do you think the mutation in Sos might alter its activity (2pts)? Sos is a Ras-GEF meaning it helps exchange ADP for ATP which activates the G Protein Ras. In this case, the mutation of SOS alters its activity by increasing its function so it constantly activates G-protein Raf through the exchanging of ADP to ATP. b) How does this mutation affect the other components of the ERK pathway (think about the components that are upstream and downstream of Sos) (2pts)? The mutation leads to increased activation of Raf, followed by phosphorylation of MEK and ERK activating them. Upstream of Sos1 is the Grb2 which will not be affected as the structure of Sos does not change and will still successfully bind to it for the pathway to be activated. c) The other five individuals with Tiger syndrome in the trial don’t have mutations in Raf or Sos. Which components of the pathway would you sequence next, and why (2pts)? We would sequence MEK and ERK next as it is downstream of Raf and Sos1. It is likely that if there exists no mutations in Sos and Raf, for the Tiger syndrome to occur there has to be some mutation in MEK or ERK. d) Some individuals with Lion syndrome do not have any mutations in Raf, but have a mutation in ERK that makes it constitutively active (it is a phosphomimic mutation that makes the protein act like it is always phosphorylated). Consider each of the drugs/therapies below and explain how they may or may not affect the Lion syndrome patients with this kind of ERK mutation (3pts). i. a Raf inhibitor: A phosphomimic ERK mutation would activate the pathway leading to increased cell proliferation. Inhibiting Raf would allow us to prevent the phosphorylation of MEK and ERK. However, the mutation causes ERK to be in the activated conformation without the signaling from Raf hence Raf inhibitor will not be an effective treatment for cancer in Lion Syndrome. ii. an antagonist for the RTK: An antagonist for RTK will interfere with the activation of RTK and will not allow the MAPK/ERK pathway to be activated. Hence, there will be no phosphorylation of ERK but in Lion Syndrome ERK has a phosphomimicking mutation which means it will be activated regardless of the pathway. Hence, an antagonist for RTK will not be an effective treatment. 4

iii. an shRNA that is designed to target ERK: shRNA is a short hairpin RNA, which leads to knock-down gene expression or silencing of target mRNA. If this occurs, ERK will be silenced or even degraded down. As a result, ERK will not be able to interact with the nucleus to produce the DNA. 5

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