Cell Signaling I: Signal Transduction and Short Term Cellular Responses | 146 470, Study notes of Cell Biology

Download Cell Signaling I: Signal Transduction and Short Term Cellular Responses | 146 470 and more Study notes Cell Biology in PDF only on Docsity! 1 ADVANCED CELL BIOLOGY// MOLECULAR BIOLOGY OF CELLS 01-146:470 + 16-148:514 •Monday 11.24.2008 (8:40-10:00) •SEC-118 • Class web page: http://lifesci.rutgers.edu/~denhardt/course/cellmolbiol.htm •Dr. Guy Werlen •Dept. of Cell Biology & Neuroscience •Nelson Bio. Labs., B333 •werlen@biology.rutgers.edu •Office hours, Friday 3:30pm-5:00 pm Molecular Cell Biology 6th Edition Chapter 15: Cell signaling I: Signal transduction and short-term cellular responses Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira - 15.1 From extracelluar signal to cellular response. - 15.2 Studying cell-surface receptors. - 15.3 Highly conserved components of intracelluar signal-transduction pathways. - 15.4 General elements of G protein-coupled receptor systems. - 15.6 G protein-coupled receptors that activate or inhibit adenylyl cyclase. - 15.7 G protein-coupled receptors that activate phospholipase C. From Extracellular signals to cellular responses From extracelluar signal to cellular response, or why signal transduction is a must 1) Synthesis and packaging of secreted molecules (ligands). 2) Exocytosis 3) Transport of ligand to target cell 4) Binding of ligand to receptor 5) Activation of intracellular signaling pathways. 6a) Short term modifications 6b) Long term modifications 7) Signaling inhibition (“feedback loop”) 8) Removal of ligand 2 Close range, or far reaching signaling Distance: several meters Distance: a few micrometers Distance: “a slap on your face” Attached ligands can also induce signaling Ligands attached on one cell can trigger signaling in an adjacent target cell (adherence molecules such as integrins) Ligand-Receptor interactions: Harmonic “groove” Essential residues on the ligand and the receptor determine a specific binding. a) In this example only 8/28 aa of the hormone that are found at the binding interface of the hormone to the receptor are contributing 85% of the binding energy (pink in the cartoon). Similarly, while several aa of the receptor binding interface are important (yellow), 2 tryptophan (trp) residues (blue) contribute most of the energy for binding growth hormones. b) Binding of growth hormone to 1 receptor molecule is followed by binding of a 2 receptor (c; purple) to the opposing side of the hormone; this involves the same set of aa on the second receptor (yellow and blue in the cartoon), but different residues on the hormone. c) Hormone-induced receptor dimerization is a common mechanism for activation of receptors and the start of signal transduction. Studying cell surface receptors Specific signaling mechanisms exist downstream of distinct receptor families, but they are all activated by ligand binding to the receptor and their activation mainly leads to gene transcription. 5 Phosphorylated nucleotide derivatives serve as second messengers Highly diffusible molecules such as cAMP or cGMP are widely used to transduce signals even if the target is localized in intracellular stores. Lipid derivatives serve as second messengers. Lipid derivatives act as second messengers at the membrane (for example, DAG) or such as IP3 translocate to particular organelles across the cytosol. DAG and IP3 are generated by the cleavage of membrane localized phosphoinositol phospate (PIP2). DAG remains in the membrane, while IP3 is released into the cytosol and activates the release of calcium, Ca2+ from storing organelles. [Ca2+]i= ~50 nM [Ca2+]e= ~1 mM [Ca2+]i= ~10 µM PLCγ PIP2 DAG IP3 Ca2+[Ca2+]i= ~500-1000 nM Intracellular Ca 2+ mobilization Intracellular calcium concentrations, [Ca2+]i are tightly regulated. In a resting cell [Ca2+]i ~ 50 nM, while ~ 1mM Ca2+is found in the extracellular environment ([Ca2+]e). Distinct organelles, such as the Golgi, the nucleus or specialized Ca2+stores (calciosomes) contain up to 10 µM Ca2+. Engagement of a receptor by a ligand induces the activation of the phospholipase PLCγ that cleaves membrane localized PIP2 into diacylglycerol (DAG) and Inositol trisphospate (IP3). DAG stays in the membrane, while IP3 diffuse through the cytosol and binds to IP3 receptors (IP3R) that are localized on the surface of Ca2+-storing organelles. The engagement of IP3R triggers the opening of Ca2+-channels on the surface of the storing organelles and a burst of Ca2+ is released into the cytosol. This in turn activates cell membrane localized Ca2+-operated-Ca2+-channels and the entry of more Ca2+ from the extracellular milieu, resulting in 10-20 fold increase of [Ca2+]i. Ca2+-homeostasis is restored by ligand-receptor complex dissociation, PLCγ inactivation and the repumping of Ca2+ into the storing organelles. Base line Thy/APC Ligand induced Ca 2+ i mobilization 0 20 40 60 80 100 120 140 160 180 200 0 60 120 180 240 300 360 420 time (sec) flu or es ce nc e in te ns ity F1 (340) F2 (380) Ratio F2min [Ca2+]I=Kd Q Q=F2min/F2max F2maxRmin Rmax Ca2+ calibration Ca Cl 2 Tx -1 00 EG TA Tr is p H 8. 4 Fura-2 Roger Y. Tsien Fluorescent Ca2+-binding dyes, such as the EGTA-derived compound Fura-2 measure changes in [Ca2+]i. Before agonist stimulation, cells are preincubated with fura-2 and the baseline of fluorescence (corresponding to homeostatic [Ca2+]I) is established. The addition of an agonist induces Ca2+ release and and increase the amount of Ca2+bound to fura-2 is indicated by an increase in the fluorescence at 340 nm (blue ligne) and a corresponding decrease of the fluorescence at 380 nm (red ligne) that corresponds to the fluorescence of free fura-2. After fluorescence recording for a certain time (~350 sec), the system is calibrated for maximum and minimum Ca2+ present in the measuring environment. [Ca2+]i are determined by using the described formula (Grynkiewicz, G., Poenie, M. & Tsien, RY. 1985. J. Biol. Chem., 260: 3440 – 3450). 6 0 100 200 300 400 500 600 0 60 120 180 240 300 360 time (sec) [C a2 + ] i( nM ) OVA 2 µM Q4 2 µM T4 2 µM Q7 2 µM G4 2 µM E1 2 µM no peptide OT-1 DP Thymocytes ([Ca2+]e = (2 mM) { {High affinity ligands Low affinity ligands OVA is an octameric peptide from Ovalbumin with the sequence SIINFEKL. OVA is specifically recognized by the MHC class I restricted TCR, OT-1. The other peptides are variants of OVA with single amino acid switches in position one, (E1), four (Q4, T4, G4), or seven (Q7). Faint lines represent Ca2+ measurements in presence of 2 mM extracellular Ca2+. Plain lines represent Ca2+ measurements in absence of extracellular Ca2+ 2 µM OVA 2 µM OVA/ Ca2+ free 30 60 90 120 150 180 210 240 270 time (sec) 0 50 100 150 200 250 300 0 [C a 2+ ]i (n M ) OVA is a high affinity ligand derived from Ovalbumin that specifically binds the OT-1 TCR. The dark colored plain lines represent Ca2+ measurements in presence of 2 mM extracellular Ca2+ , while the faint doted light colored lines represent Ca2+ measurements in absence of extracellular Ca2+. Blocking phospholipase γ (PLCγ), inhibits the formation of IP3 and Ca2+release. Blocking the Ca2+ channel, inhibits Ca2+ repumping into the Ca2+ stores or the extracellular medium. The Src kinase inhibitor, blocks Ca2+ release or intake mechanisms that are regulated by Src-like tyrosine kinases. PLCγ blocker 1 µM + OVA PLCγ blocker 100µM + OVA PLCγ blocker 10 µM + OVA PLCγ blocker 1 µM + OVA/ Ca2+free Channel blocker 10 µM + OVA Src kinase blocker 10 µM + OVA Channel blocker 10 µM + OVA/ Ca2+free Src kinase blocker 10 µM + OVA/Ca2+free Signal transduction = signal amplification The interplay of second messengers and signaling proteins amplifies the signal and accounts for signal specificity. In this example, binding of a single epinephrine molecule to one receptor induces the synthesis of a large number of cAMP molecules, the first level of amplification. Four molecules of cAMP activate 2 molecules of protein kinase A (PKA), but each activated PKA phosphorylates multiple target molecules. This second level of amplification may involve several sequential reactions in which the product of one reaction activates the enzyme catalyzing the next reaction. The more steps in such a cascade, the greater the signal amplification. General elements of G protein-coupled receptor (GPCR) systems 7 GPCR are hepta-(membrane)spanning molecules All GPCR have seven trans-membrane domains (H1-H7),four extracellular segments (E1-E4), and four cytosolic regions (C1-C4). Specific residues of a ligand, such as Epinephrine engage particular residues of the GPCR (β2-adrenergic receptor in the case of epinephrine). The G-Protein GPCR activate trimeric G-proteins The Gα and Gβγ subunits of trimeric G proteins are thetered to the membrane by covalently attached lipid molecules (wiggly black lines). In the inactive “off” state Gα binds GDP (similar to a small G protein), upon activation of the receptor associated to the trimeric G protein, GDP is exchanged for GTP and Gα undergoes a conformational change. The Gβγ subunits are regulatory (in some instances they bind the receptor). GPCR associate to trimeric G proteins Intracellular trimeric G proteins transduce the signal generated by engagement of GPCR. Trimeric G proteins are composed of the catalytic subunit Gα that binds GTP or GDP as well as the regulatory subunits, Gβ and Gγ. The long C3 loop of GPCRs interacts with G proteins Expression of chimeric constructs of GPCRs has characterized their C3 loop as critical in binding downstream G proteins. Xenopus oocytes that normally do not express adrenergic receptors, were microinjected with mRNA encoding α2-adrenergic, β2-adrenergic or chimeric αβ-receptors. The adenylyl cyclase activity of each oocyte was measured in response to epinephrine, which determined whether the expressed receptor was binding to the stimulatory, Gαs or inibitory, Gαi type of oocyte G proteins. By comparing chimeras 1 (interacts with Gαs) and 2 (interacts with GαI), it was possible to determine that the C3 loop (yellow) between the α helices 5 and 6 determines the specificity of the binding G protein. 10 PLC activation generates DAG and IP3 Phospholipase C (PLC) cleaves PIP2 into DAG and IP3. DAG stays within the membrane’s cytosolic leaflet, while IP3 is released into the cytosolic compartment. Intracellular Ca2+ acts as second messenger Both IP3 and DAG acts as second messengers. 6) DAG activates signaling molecules such as protein kinase C (PKC), while 3) IP3 binds to an IP3 receptor that is expressed on the cytosolic leaflets of organelle membranes and 4) induces calcium channels to open and release Ca2+. 5) Ca2+ released into the cytosolic compartment will 7) activate Ca2+-dependent proteins such as PKC. The depletion of Ca2+ from its storage organelles will induce a channel operated entry from extracellular Ca2+ that will replenish the organelles. Ca2+ as a regulator of cell responses NO/cGMP control arterial smooth muscle relaxation 1-4) Nitric oxide (NO) is synthesized in endothelial cells in response to acetylcholine and the subsequent elevation of Ca2+. 5-6) NO diffuse to nearby smooth muscle cells and triggers cGMP production via its binding to NO receptors. 7-8) cGMP activates protein kinase G (PKG) and induces the relaxation of the smooth muscles. 11 Interaction of signaling pathways specify cell responses The interplay of activation and inhibition signaling pathways eventually account for the specificity in cell responses. A) Neuronal stimulation of striated muscle cells or epinephrine binding to β-adrenergic receptors on their surfaces leads to increased cytosolic Ca2+ or cAMP, respectively. The key regulatory enzyme glycogen phosphorylase kinase (GPK) is activated by Ca2+ ions and by PKA. B) In liver cells, hormonal stimulation of β-adrenergic receptors leads to increased cytosolic cAMP as well as DAG and IP3. IP3increases [Ca2+ ]i and glycogen degradation, while DAG activates PKC that will block glycogen synthase in synergy to PKA.