Answer Key: Signal Transduction

BIOL230W Week 7 – Signal Transduction This week we will be focusing on a different type of information flow in cells: signal transduction . While the central dogma helps explain the flow of genetic information from the DNA to protein, signal transduction (also calledcell signaling ) allows cells to respond to environmental cues, communicate with each other and undergo normal growth and development. Proteins and other small molecules play an important role in this process . This week we will be learning the components of signaling pathways and how to read visual representations of these pathways. Day 1 Learning Objectives 1. Describe the general process of signaling (including signals, receptors, second messengers and final target/effector). 2. Define protein conformation changes and how conformation changes result in changes in function. 3. Compare and contrast the characteristics of GPCRs, RTKs, intracellular androgen receptors. 4. Describe the role of G-proteins in signaling. 5. Use legends to integrate information about the function of proteins (Worksheet 9). 6. Compare and contrast peptide motifs and structural motifs. Small Group Activity: Summarize Parts of a Pathway (5 minutes) In your own words (or with a neighbor), summarize from the exploration homework each part of a signaling pathway. Try to include information about the structure and function of each component. 1. Ligand: a chemical signal (can come from cell or environment) 2. Receptor: detect a ligand to induce a physical change in itself (usually change in protein conformation/3-dimensional shape) 3. Effector: Proteins altered by the pathway of transduction, separate (signal received by receptor, causes change in conformation of other proteins in the cell outside of the receptor) 4. Secondary Messengers: non-proteins, released in response to effector Mini-Lecture: Parts of a Pathway Receptors 5. How does binding of a ligand to a receptor alter the receptor? Ligand binds to the part of the receptor that extends outside of the membrane (since receptors are integral membrane proteins) and alters the conformation (changes it shape). Ligand binds to specifically shaped region on receptor, and signal from ligand binding transfers across the membrane. 6. What protein components make up a GPCR? Transmembrane proteins that are integral (7), extracellular ligand-bind region, and intracellular Gprotein coupled domain (bound to G-proteins in membrane) Small Group Activity: Structure of GPCR Receptor 1 BIOL230W Week 7 – Signal Transduction The image below represents the protein structure of the receptor part of a GPCR. Each small circle represents one amino acid. Modifications to the amino acids are shown in the top two boxes. Peptide (not structural) motifs are shown in the bottom box. Peptide motifs are short, conserved sequences of amino acids (like DNA consensus sequences) that serve a function. Use this image to answer the questions. 7. Highlight the regions of this GPCR important for ligand binding. 8. Highlight the region of this GPCR important for G-protein binding. 9. How many alpha helices of the GPR pass through the membrane? 7 10. Does this GPCR contain any lipidated peptide motifs? Yes 11. What do you think would happen if the S/T, X, N motif was mutated in this GPCR? The GPCR would not fold correctly and would not be brought to the correct place 2 BIOL230W Week 7 – Signal Transduction Day 2 Learning Objectives 1. Describe the general process of signaling (including signals, receptors, second messengers and final target/effector). (Continued from Day 1) 2. Define protein conformation changes and how conformation changes result in changes in function. (Continued from Day 1) 3. Compare and contrast the characteristics of GPCRs, RTKs, intracellular androgen receptors. (Continued from Day 1) 4. Describe how cAMP and DAG/IP3 + Ca2+ are synthesized in the cell. 5. Explain how receptor activation leads to the production of second messengers. 6. Describe the function of the major effector molecules (kinases and phosphatases) and how they work together. Mini-Lecture: Parts of a Pathway Receptors cont. 12. What are two distinguishing features about receptor tyrosine kinases? Ligand binds on extracellular part of cell; 2 parts of receptor comes together. Second Messengers 13. What enzyme is activated to produce cAMP? Adenylyl cyclase 14. What is one possible effector (target) activated by cAMP? Protein kinase A (PKA) Small Group Activity: CFTR Conformation Changes Use the image and caption below to answer the following questions. The impact of PKA-mediated phosphorylation on CFTR channel. The cystic fibrosis transmembrane conductance regulator (CFTR) structure consists of two membrane spanning domains, two nucleotide-binding domains (NBD1 and NBD2), and one cytoplasmic regulatory (R) domain. The R domain represents a critical site of channel regulation due to its enrichment in protein kinase A (PKA) peptide motifs (consensus sequence, with multiple serines and threonines as phosphorylation targets). There is an additional phosphorylation site in the regulatory insertion (RI) segment of NBD1. In the closed CFTR state (left channel), the NBD1 interacts with the R 3 BIOL230W Week 7 – Signal Transduction domain creating steric hindrances which prevent it from dimerization with NBD2. Upon PKAdependent phosphorylation (right channel), large conformational changes occur that decrease this interaction leading to the release of the R region from its inhibitory position and allow NBD dimerization, ultimately ATP binding and CFTR-mediated chloride secretion. Amino acids targeted for phosphorylation are shown in red and yellow. From Della Sala, A., Prono, G., Hirsch, E., & Ghigo, A. (2021). Role of Protein Kinase A-Mediated Phosphorylation in CFTR Channel Activity Regulation. Frontiers in Physiology, 12. 15. What domain of CFTR is primarily targeted for phosphorylation? Cytoplasmic Regulatory Domain 16. What two amino acids are commonly phosphorylated (shown in this example)? Serine and Threonine 17. What two domains of CFTR must dimerize to allow the diffusion of Cl-? NBD1 and NBD2 18. When the CFTR is in the conformation on the right, which direction will osmosis occur? From into the cell to outside of the cell Mini-Lecture: Parts of a Pathway Second Messengers (cont.) 19. What enzyme is activated to produce IP DAG? 3 and Phospholipase C Effectors 20. Compare and contrast kinases and phosphatases. Kinases add phosphates to proteins, whereas phosphatases remove phosphates from proteins. 21. What type of macromolecule are effectors? Proteins Day 3 Learning Objectives 1. Interpret example signaling pathways from ligand to final effector. 2. Identify representative molecules from the categories (signals, receptors, second messengers, effectors, final targets) that we discussed in class of example pathways. 3. Identifying "upstream" vs. "downstream" parts of a path and how they relate to function. 4. Determining the result of a pathway if specific effectors (like kinases or phosphatases) are inhibited or enhanced. 5. Evaluate the final cellular change of a pathway Practice DAY!!! 4 BIOL230W Week 7 – Signal Transduction Learn to read signaling pathways. Make sure to look over the whole diagram and carefully read the captions. Small Group Activity: Inflammation Pathway Role of cytosolic phospholipase A2 (cPLA2) in the activation of inflammation. GPCR activation results in IP3 and DAG production. Calcium binding to the C2 domain of a phosphorylated cPLA2 triggers membrane localization of cPLA2 and subsequent hydrolytic release of arachidonic acid from phospholipids, a precursor to inflammatory prostaglandins. COX synthesizes prostaglandins that mediate inflammation using the freed arachidonic acid. COX function is blocked by aspirin, ibuprofen, and other anti-inflammatory drugs. 22. What type of receptor is utilized in this pathway? How do you know? GCPR – GCPR activation leads to DAG/IP3 production 23. What are the second messengers? DAG, IP3, Ca2+ 5 BIOL230W Week 7 – Signal Transduction 24. What is the target protein of this pathway that is activated by ligand binding (hint-COX is always active as long as there is arachidonic acid present)? cPLA2 25. What is the function of cPLA2? What type of an enzyme is cPLA2? Inflammation activation 26. What is the general cellular response of this pathway Inflammation Small Group Activity: HIPPO Pathway Hippo signaling is a conserved pathway that controls cell proliferation, apoptosis (cell death), and organ size in response to cell density. At low cell density (Figure 1, on the left), transcription co-activators YAP and TAZ enter the nucleus and induce expression of genes that favor cell growth and proliferation. As cell density increases (figure 2 on the right), interactions between membrane bound FAT4 receptors and DCHS1/2 on neighboring cells trigger activation of cytoplasmic kinases Mst1/2 and LATS1/2. These kinases phosphorylate TAZ/YAP and prevent them from entering the nucleus, thereby reducing gene expression. 27. What is the ligand in this pathway? DCHS 1/2 28. What is the receptor in this pathway? FAT4 6 BIOL230W Week 7 – Signal Transduction 29. What are the target proteins in this pathway? What type of proteins are these (we’ve seen these in class during Week 3)? 30. What happens when the target proteins are phosphorylated? 31. What is the ultimate cellular response if this pathway is activated? 7