7.06_2004_PS2 - 7.06 Spring 2004 PS 2 1 of 4 7.06 Problem...

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Unformatted text preview: 7.06 Spring 2004 PS 2 1 of 4 7.06 Problem Set 2 1) You have started working in lab that investigates signaling pathways downstream of TGFß receptors. One of the senior graduate students has been working on a screen for novel TGFß’s and has one that she thinks might interest you—she calls it TGFß57. She’s already cloned the gene so you can make pure TGFß57. You decide to take up the challenge of characterizing this new growth factor. a) You would first like to find some high affinity receptors that bind your novel growth factor. Design an experiment to identify the cell-surface TGFß57 receptors. b) You would now like to investigate the binding properties of TGFß57. You decide to look at the maximal physiological response of TGFß57R to TGFß57. Based on the graph below determine the Kd (in relative concentration) of TGFß57 binding, the percentage of receptors occupied at 50% of maximal physiological response, and the percentage of maximal physiological response when half the receptors are occupied. What does this tell you about the percentage of bound receptors needed to achieve maximal response? *** NOTE: y-axis label: Fraction of maximum cellular response c) You would now like to determine the binding affinity of TGFß57 for the TGFß57 receptor that you have been working with. Design an experiment to measure the Kd of TGFß57 binding high- affinity receptors in a cultures fibroblast cell line. Explain how you would deal with TGFß57 binding nonspecifically to low-affinity receptors. d) Using the specific binding curve below, determine the Kd of TGFß57 for high-affinity receptors. Also, find the total number of receptors per cell. *** NOTE: y-axis label: [I125] TGFb-57 Bound (molecules per cell) 7.06 Spring 2004 PS 2 2 of 4 e) You’d now like to determine the sensitivity of these cells to TGFß57. You know that the Kd for binding TGFß57 to its receptor is about 1X10-8 and you know there are a total of 19,000 receptors per cell. You have previously determined that only 13% of the 19,000 receptors must be bound to obtain the maximal cellular response. Determine the TGFß57 concentration needed to induce the maximal response. What would be the concentration of TGFß57 required to obtain maximal response if there were only 5000 receptors per cell, what is the fold difference compared to the normal concerntration you just calculated, and would this make the cell more or less sensitive to TGFß57? Why might a cell want to regulate the number of receptors to a given external signal? f) If TGFß57 binding to its receptor had occurred with a low affinity (a Kd higher than 1X-7M), you couldn’t have measured the Kd by performing the binding assay from above. How could you have determined the Kd if this had been the case? 2. A colleague in your lab discovers that the binding of TGFb57 to its receptor turns on expression of a gene that inhibits cell growth, SloGro. SloGro is frequently mutated in cancers, and you suspect that components involved in the TGFb57 signaling pathway might also be relevant to cancer research. You decide to set up a cell culture screen to identify proteins involved in TGFß57 signaling. Your screen is a success, and you isolate a number of cell lines that have mutations in the TGRb57 signaling pathway. A) Explain how the following mutations would affect TGFb57 signaling. Assume that the basic components of the b57 pathway are identical to the canonical TGFb signaling pathway. i)The MH1 domain of Smad 3 is missing. 7.06 Spring 2004 PS 2 3 of 4 ii)R1 has a truncated cytoplasmic tail and cannot be phosphorylated. iii) Smad 7 is constitutively active. iv) You also isolate a version of Smad3 that cannot bind DNA, and find that this mutant version exerts a dominant negative effect on the TGFß57 pathway. Give one possible mechanistic explanation for how mutant Smad3 could act dominant negatively on the pathway. 3) Epineprhine increases the concentration of glucose 1-phosphate in wild type liver cells. Explain how the following mutations would affect the glucose 1-phosphate concentration within an epinephrine stimulated liver cell. i) The cell contains a PKA with a nulceotide binding site that is unable to bind cAMP. ii) The cell contains an unphosphorylatable version of Inhibitor of Phosphoprotein Phosphatase. iii) The cell contains a mutant version of Gsa that cannot hydrolyze GTP . 4) a) Given the following values, calculate the electrochemical equilibrium potential of each ion (F=23, 062cal/(mol V), R = 1.987 cal/K mol, T = 293K). K+out = 3 mM Na+out = 117 mM Cl-out = 120 mM K+in = 90 mM Na+in = 30 mM Cl-in = 4 mM b) What does the electrochemical equilibrium potential reflect/what is true at the electrochemical equilibrium potential? c) If one reduces the magnitude of the K+ concentration gradient across the cell, one will increase/decrease (circle one) the equilibrium potential. Why? d) Assume that the resting membrane of a cell is determined primarily by the equilibrium potential of potassium. If you insert an electrode into this cell and clamp the membrane voltage to +20 mV, would K+ be moving into or out of the cell through the potassium channels (K+out = 3 mM, K+in = 90 mM)? e) You isolate cells that have only K+ channels. You also have a drug that causes this channel to open. With a voltage clamp apparatus, you find, as expected, that in the presence of the drug the membrane potential is dependent on the concentration of extracellular K+ used in the experiment. 7.06 Spring 2004 PS 2 4 of 4 At one concentration of K+ of 5 mM, you find that the membrane potential is -80.5 mV. From this information, calculate the intracellular K+ concentration. 5) You are doing a summer internship in a lab that studies a recently discovered receptor called RKR. It is a G-protein coupled receptor that is expressed in kidney cells and found to be mutated in patients with a kidney disease. You sequence the receptor in 100 patients and find that the mutations fall into two classes, A and B. Unlike wildtype RKR, both mutants fail to increase cellular levels of cAMP. (i)The ligand for the RKR receptor is called MC. How could you find out if the mutant receptors are expressed on the surface of kidney cells? (ii)You find that mutant A is localized to the membrane at approximately the same levels as wildtype but mutant B has very low levels of RKR receptor on the surface. Mutation A lies in the conserved C3 loop, which is known to be important for binding and activation of the Ga subunit. How could you determine whether Ga becomes activated in cells expressing mutant A? (iii) While trying to understand the basis for mutant B you look at receptor localization in the presence and absence of ligand using the GFP tagged versions of the receptors from part (i). What do you expect to see in the wildtype cells after extended exposure (30mins) to ligand? In the cells expressing mutant B the receptor is found to localize predominantly to the cytoplasm in the presence or absence of ligand. How could you explain this? How could you demonstrate this experimentally? (iv) The RKR receptor has a conserved motif in the cytoplasmic portion of the protein that contains Ser-Ser-Thr. You want to show that if you prevent the constitutive b-arrestin mediated endocytosis of the mutant B version of the RKR receptor that it will then be capable of signaling Your advisor suggests that you mutate all three residues to alanine in the mutant receptor? Why does he choose these three residues? Why does he suggest mutating them to alanine? ...
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