MCB*4010 Midterm Exam / Winter 2008 Name: ID: Instructions: Answer all 4 questions. The number of marks for each question indicates how many points you need to provide. Write your answers in point form, providing as much detail as possible. Q1A 8 Q1B 6 Q2A 8 Q2B 6 Q3A 8 Q3B 3 Q3C 3 Q3D 1 Q4A 8 Q4B 6 TOTAL 57 % /40
Question 1 A. [8 marks] Describe the steps from the binding of epinephrine to its receptor and the activation of PKC. Include a description of how hormone binding to the GPCR leads to changes in the conformation and activity of affected G-protein and effector molecules. 1. Epinephrine binds to alpha2-adrenergic receptor (trimeric G-protein coupled receptor) altering the conformation of the cytosolic domain 2. Gqα subunit of trimeric G protein binds to activated receptor. This causes release of GDP and binding of GTP to Gqα 3. Gqα -GTP dissociates from Gβ and Gγ subunits 4. Gqα -GTP binds to its effector, phospholipase C. Interaction of switch domain of Gqα -GTP with phospholipase C holds phospholipase C in an active conformation 5. Phospholipase C cleaves PIP2 (PI 4.5-bisphosphate) to produce DAG (1,2-diacyclglycerol) and IP3 (inositol 1,4,5-trisphosphate) 6. IP3 binds to an ER calcium channel opening the channel and allowing calcium to enter the cytoplasm 7. DAG diffuses through the plasma membrane and interacts with PKC. Binding of DAG and calcium to PKC results in its activation. 8. PKC is released and phosphorylates specific transcription factors in the nucleus. B. [6 marks] Light perception is signaled through G-protein coupled receptors. Name each of the following components of the signal transduction cascade: receptor: Gα-protein component: effector molecule: Rhodopsin Gtα (transducin) cgmp phosphodiesterase 2 nd messenger: cgmp target of 2 nd messenger: effect of 2 nd messenger on target: sodium channels channel opens
Question 2 A. [8 marks] Using the epidermal growth factor receptor as an example, describe the steps from ligand/receptor interaction to activation of a kinase-signaling pathway leading to the activation of Ras. 1. EGF-R monomers are membrane-spanning proteins with a ligand-binding domain located outside of the cell and a tyrosine kinase domain within the cell. The EGF monomer binds to a receptor altering the conformation of the extra-cellular domain allowing dimerization. 2. The dimerized EGF receptor brings the associated weakly active tyrosine kinase domains in close proximity resulting in trans-phosphorylation. 3. Activated kinase domains cross phosphorylate additional tyrosine residues. 4. The Grb2 adaptor protein (which contains SH2 and SH3 domains) binds to phosphotyrosine residues on the activated receptor through an interaction involving its SH2 domain 5. The guanine-exchange factor (GEF) Sos binds to SH3 domains on Grb2 localizing it to the activated receptor 6. Inactive Ras (Ras-GDP) is bound to the plasma membrane through its C-terminal farnesyl anchor. 7. Sos interacts with Ras-GDP stimulating GDP release by altering the conformation of the two switch domains. 8. GTP binds to Ras resulting in its activation. B. [6 marks] Lowenstein et al. (1992) delineated two distinct domains on GRB2 that were important in growth factor signaling. What are the two types of domains that make up the GRB2 protein? What proteins do each of these domains interact with? What residues on these proteins do the domains recognize? SH2 domain binds to phosphotyrosine residue on activated growth factor receptors SH3 domain binds to proline-rich region on SOS
Question 3 A. [8 marks] Ras activation signals through a MAPK cascade leading to changes in gene expression. How does Ras regulate the activity of the proximal MAPKKK and how is this signal transmitted through MAPKK to MAPK? Fill in the blanks below to indicate the name of the kinase and the residues (which type of amino acid) that they phosphorylate on their target proteins. Note that some blanks will contain the same answer as some other blanks and that in some cases more than one residue will be phosphorylated. Raf is a MAPKKK that phosphorylates serine residues on MEK. MEK is a MAPKK that phosphorylates threonine and tyrosine residues on ERK ERK is a MAPK that phosphorylates serine and threonine residues on specific transcription factors B. [3 marks] How does phosphorylation of MAPK by MAPKK lead to MAPK activation? Describe in term of changes in the structure of the MAPK. Catalytic site in inactive ERK is blocked by activation lip MEK phosphorylates threonine and tyrosine residues in activation lip of ERK Position of activation lip is altered opening the catalytic site allowing binding of ATP and substrates C. [3 marks] Describe how Ras activates Raf. 1. In unstimulated cells Raf exists in an inactive conformation with its N-terminal regulatory domain bound to its C-terminal kinase domain. 2. This conformation is stabilized by binding of 14-3-3 proteins to the phosphoserine residues in the N and C terminal domain. 3. Binding of Ras-GTP to the N-terminal regulatory domain of Raf induces a conformational change that results in release of 14-3-3 proteins and activation of Raf kinase activity. Active Raf dissociates from Ras following GTP hydrolysis and phosphorylates MEK in the cytosol. D. [1 mark] Saccharomyces cerevisiae has five distinct MAPK pathways that respond to different signals and produce different responses although they share some of the same kinases. What type of signaling molecule is used to direct the signals to the proper response? Pathway specific scaffold proteins orient the kinases for a specific pathway in their proper sequence.
Question 4 A. [8 marks] Describe the steps leading from TGFβ binding to its receptor to the activation of gene expression. 1. Receptor composed of 2RI and 2RII subunits that exist as unligated dimers in unstimulated cells. TGFβ binding to RI and RII causes receptor multimerization 2. RII dimer has constitutively active kinase activity (autophosphorylated) 3. RII phosphorylates serine residues in GS domain of RI 4. Phosphorylation of GS domain changes conformation of RI, which allows Smad binding. 5. SMADs are latent cytoplasmic transcription factors. N-terminal MH1 domain contains DNA binding domain and NLS. C-terminal MH2 domain binds RI and masks MH1 domain. 6. Phosphorylation of serine residues on SMAD by activated RI alters conformation of SMAD resulting in exposure of the MH1 NLS 7. A dimer of phosphorylated SMAD3 or SMAD2 binds SMAD4 and together with importin β is translocated into the nucleus 8. Importin is released and the SMAD-coSMAD complex binds promoters and activates transcription in cooperation with promoter-specific transcription factors. B. [6 marks] TGFβ receptors and cytokine receptors have the following characteristics: TGFβ receptors have an intrinsic kinase activity that phosphorylates the cytosolic SMAD transcription factors on serine residues. Cytokine receptors have an associated JAK kinase that phosphorylates the cytosolic STAT transcription factors on tyrosine residues.