Lecture 17 - Cell Signaling - Bio 220 PDF

Bio 220 Lecture 17 Signal Transduction- G proteins Oct 29, 2024  Key Concepts 1. Cell-Cell Signaling: ancient, varied for different functions, cooperative, importance of cell context, second messengers molecular switches, cascade amplification, keeping & turning pathway off...

Bio 220 Lecture 17 Signal Transduction- G proteins Oct 29, 2024  Key Concepts 1. Cell-Cell Signaling: ancient, varied for different functions, cooperative, importance of cell context, second messengers molecular switches, cascade amplification, keeping & turning pathway off 2. G-Protein Linked/Coupled Receptors (GPCR): large family, trimeric G proteins, turning off G proteins, adenylyl cyclase/cAMP, PKA, IP3, phospholipase C-b, Ca2+ mediated signaling, CaM-Kinase, olfaction  Cell Signaling What are some examples? What is the function? 3  1. Cell-Cell Signaling: Important Principles Proteins, peptides, amino acids nucleotides, steroids, retinoids, >1500 dissolved gases (NO), light, receptors touch, heat (many released by in humans exocytosis, some transmembrane) effector proteins a) Origins of Signaling- Ancient Signaling between cells is ancient. Our own cell-cell signaling systems almost certainly evolved from cellular signaling systems from our unicellular ancestors. e.g., S. cerevisiae mating factor, small peptides Formation of the Shmoo for mating. Bacterial Films 6  K+ efflux changes transmembrane voltage, and leads to K+ ion release in neighboring cells from Nature 2015, “Electrical signaling goes bacterial” 7  cAMP in Dictyostelium discoideum starvation The signal transduction pathways in which Dictyostelium chemotax and leukocyotes move to sites of infection are the same and appear to be conserved. 9 b) Reception of signal Two main locations for receptors Most cell-cell signaling pathways (Ion channel receptors, G Protein-coupled, Enzyme-coupled) Nuclear hormone Receptor signaling nitric oxide (NO gas) 5-10 sec 1/2 life smooth muscle nerves 5-10 sec half life Guanylyl cyclase is both receptor & signaling protein Steroid and thyroid hormone molecules bind to nuclear hormone receptors (receptors-act as both receptors and effectors) small & hydrophobic **stay in blood stream for hours to days 278 nuclear hormone receptors in C. elegans 48 in humans 21 in Flies c) Forms of intercellular signaling A&B Short Used often Range in development e.g. Immune system, Notch e.g. Most develop. signals C&D Long Important for Range large organisms/ homeostasis  autocrine Lodish et al.,, Molecular Cell Biology 14  Slower, must work at low concentrations, receptors high affinity. Response dictated by cells that express/have the receptors Figure 15-5a Molecular Biology of the Cell (© Garland Science 2008)  Faster 100m/sec receptors low affinity Specificity achieved by synaptic connection (all of these can use the same neuro- transmitter/ receptor) Figure 15-5b Molecular Biology of the Cell (© Garland Science 2008)  signals can have a fast effect or a slow one Figure 15-6 Molecular Biology of the Cell (© Garland Science 2008) d) Cell Response to Signals Dictated by: The receptors it expresses and The intracellular context of the cell the signals it receives In development this often involves different complements of transcription factors present in different cells e) Intracellular signaling  Not a typical pathway! Just illustrates Signaling complex, binds potential places for regulation. together groups of interacting proteins (scaffolds & adaptors) a form of coincidence detection How does this compare to nuclear hormone pathway?  Network of intracellular proteins: Some of these intracellular signaling proteins act as molecular switches: Trimeric & Monomeric G proteins Can be set to allow sensitivity to signal strength, and facilitates signal amplification Signaling complex, binds together groups of interacting proteins (scaffolds & adaptors)  Modular interaction domains mediate interactions between proteins SH2 = Src homology 2 domain (binds phosphorylated tyrosine) SH3 = Src homology 3 domain (binds proline-rich regions) PTB = phosphotyrosine-binding domain (binds phosphorylated tyrosine) PH = Pleckstrin homology domain (phosphoinositides) cluster receptors/signaling through a type of phase-shift transit Creates a concentrated signaling microenvironment 24  Most cell surface receptors relay signals via small molecules (second messengers) Second Messengers: generated in large numbers in response to receptor activation and diffuse rapidly. e.g. cyclic AMP, Ca2+ , diacylglycerol (why is this important?) Earl Sutherland, Vanderbilt Epinepherine signals through a molecule he discovered, cAMP, coined term “second messenger” It increases heart rate and stroke volume, dilates the pupils, and constricts arterioles in the skin and gut while dilating arterioles in leg muscles. It elevates the blood sugar level by increasing conversion of glycogen to glucose in the liver Spreads the signal to other parts of the cell and amplifies signal one epinephrine molecule can trigger production of one hundred million glucose-1-p molecules f) pathway activation is tightly regulated 1. When ligands are not present, pathways actively turn themselves off. Example: Hedgehog Pathway Pathway off Pathway on g) Important for cells to desensitize to signals--can happen at all steps in signal transduction pathway (e.g. Hedgehog pathway & cancer).  1. 2. Patched Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened upregulated, Can 1. James K. Chen, Jussi Taipale, Michael K. Cooper, andturn off Philip A. 1 Beachy How might this pathway Pathway. be mutated in cancer? e.g. Basal Cell Carcinoma One of the target genes in some most common cancer, 93% cells of Ci is Patched, a negative Have defect in…….. regulator of Hh signaling What gene would you target to treat this cancer? Cyclopamine wild corn lily Text 31 32  2. G Protein-Linked/Coupled Receptors All cross the membrane 7 times. While they mediate many functions, and are activated by diverse ligands, all appear to operate in a similar way. 34 a) G protein-coupled receptor (GPCR) signaling (perspective): --largest family of cell surface receptors/largest gene family period (~800 genes). Involved in all types of stimulus response pathways, everything from intercellular communication to physiological senses (site, smell and taste). Examples: --Opsins (both rhodopsin (b/w, night vision) and cone opsins (color vision). --Olfactory receptors. --Brain receptors for serotonin, dopamine, GABA, glutamate (behavior and mood regulation). --Chemokine receptors in immune system functioning (chemotaxis & histamine/cancer). --Autonomic nervous system. Control blood pressure, heart rate, digestion. 1. GPCRs mediate response to an enormous diversity of signaling molecules…hormones, photons, neurotransmitters…in forms of peptides, derivatives of amino acids, nucleotides and fatty acids. 2. GPCRs involved in many pathological conditions, are targets of approximately 25-30% of all drugs. 3. Not recognized, however, as one of the seven major signal transduction pathways involved in cell fate decisions during development and stem cell differentiation. These are: Wnt TGF-b Hedgehog (Hh) Receptor tyrosine kinase (RTK) Nuclear receptor Jak/STAT Notch b) Signal through trimeric (or large) G proteins (a molecular switch) Relay signals from GPCR receptors. Functionally couple receptors to enzyme and channel effectors. Human genome encodes 21 a, 6 b , and 12 g genes. They can combine in various ways that form five groups: Gs (stimulatory) activates adenylyl (adenyl/adenylate) cyclase to increase cAMP synthesis (increased cAMP) Gi (inhibitory) inhibits adenylyl cyclase (decreases cAMP) Golf (olfactory) couples to olfactory receptors (increases cAMP) Gt (transducin) transduces visual signals in conjunction with rhodopsin (phosphodiesterase activated, decreases cGMP levels) Gq stimulates phopholipase C-b (IP3 and DAG increased) G12/13 regulates cytoskeleton, cell junctions, and other processes (activates Rho GTPase) Previously we have discussed monomeric (or small). Include Raf/Rab family members in vesicular traffic and Rho/Rac in actin dynamics, and we will talk about Ras in receptor tyrosine kinase signaling. Receptor activation results in change in conformation that results in release of GDP from a-subunit and binding to GTP What does receptor remind you of? alpha subunit often associates with an RGS (regulator of G protein signaling) protein to enhance hydrolysis. 25 different RGS proteins. Analogous to……. c) Importance of turning off trimeric G proteins: Cholera is a water-borne disease caused by the bacterium Vibrio cholerae, which is typically ingested by drinking contaminated water. The cholera toxin locks the trimeric Gs a-subunit in the active state, so high levels of cAMP persist. One of the most rapidly fatal illness known (can die within 3 hours of symptoms onset). Cholera produces potentially lethal dehydration through a pathway that involves the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC transporter. Cystic fibrosis transmembrane conductance regulator (CFTR) ABC transporter, most diverse family of transporters known, most specialized for export. MDR proteins, one exports yeast mating factor Normal CF patient Mucus not hydrated. Thus, get bacterial growth. Cl- movement provides driving force for Na2+ and ultimately water into the lumen of the lungs (& gut) G protein on cAMP activation of PKA activation of CFTR chloride water(diarrhea) Mutations in CFTR are common--perhaps selected for by cholera toxin CF is one of the most common fatal inherited diseases (4% of people of European descent carry one mutant allele). The most common mutation in CFTR is estimated to be up to 50,000 years old. Other common autosomal recessive diseases such as sickle cell anemia have been found to protect carriers from other diseases, a concept known as heterozygote advantage. Cholera toxin requires normal host CFTR proteins to function properly, and it has been hypothesized that carriers of mutant CFTR genes benefited from resistance to cholera and other causes of diarrhea. d) adenylyl cyclase/cAMP and PKA One of the main targets of Gs is the enzyme adenylyl cyclase. A transmembrane enzyme with its catalytic side on the cytosolic side of the plasma membrane. Enzyme activation causes 10-20 fold increase in cyclic AMP concentrations (usually kept very low ~10-7M), but these are rapidly reduced by cAMP phosphodiesterase (PDE). Inhibitory G-Protein (Gi), inhibits adenylyl cyclase. Response different in each cell. Substrates differ in different cell types  In most cells, cyclic AMP exerts its effects through activation of cyclic-AMP-dependent protein kinase/Protein kinase A (PKA). PKA phosphorylates other proteins on serine or threonine amino acids. Available substrates differ in different cells, partly responsible for different responses to cAMP. Often anchored to specific subcellular compartment of cell to localize kinase Many different targets  Some responses to PKA Phosphorylation are rapid (CFTR seconds): Ezrin = PKA-anchoring protein (AKAP), at least 50 different types PKA anchored to localize activity Others, such as transcriptional regulation take much longer, and can have longer lasting consequences. CREB proteins regulate many processes. One of the more interesting is long term memory. Shutting off the effects of PKA: Serine/threonine phosphoprotein phosphatases (four main groups (I, IIA, IIB, IIC). Protein phosphatase I-reverses most phosphorylations catalyzed by PKA. e) Trimeric G protein activation of inositol phospholipid signaling (another second messenger). Instead of cAMP, many G protein-linked receptors (those associated with Gq), transduce signal by activating the plasma- membrane-bound enzyme phospholipase C-b (PLCb).  DAG Many target proteins PKC recruited to membrane, activated by Ca2+, Phosphatidyl serine & DAG. IP3--small water soluble molecule that diffuses rapidly, causes release of Ca2+ from intracellular stores (removed by a phosphatase that converts it to IP2 and kinase that converts it to IP4) DAG--remains in membrane, activates protein kinase C (PKC) f) Ca2+ --a common Second Messenger (where have we seen?) Levels kept very low in cell  Ca2+ Released into cell: 1. Voltage-gated Ca2+ channels in plasma membrane 2. IP3-gated Ca2+ channels in ER 3. Ryanodine receptors-amplify calcium signals-stimulated by calcium release in Sarcoplasmic Reticulum and ER. 57  Double imaging of phase contrast and intracellular Ca2+ concentration during fertilization http://www.molbiolcell.org/cgi/content/full/9/7/1609 Ca2+ receptors inhibited by high conc of Ca2+ 59 Increasing Ca2+ initially causes both IP3 and Ryanodine receptors to release more Ca2+, but higher levels of Ca2+ then inhibits their function. As a result, waves and oscillations can form. Ca2+ oscillations in a liver cell- cell response is distinct to different oscillations…depends on Ca2+ sensing proteins Frequency of oscillations reflects strength of signal Calcium Transducers: Ca2+ binding proteins transduce cytosolic Ca2+ signal Which one have we already seen? Calmodulin: undergoes conformational change after 2 or more calcium ions bind. Protein displays sigmoidal response to calcium. Can then bind target proteins Activity of CaM-kinase II can act as a frequency decoder to different Ca2+ oscillations Figure 15-45 Molecular Biology of the Cell (© Garland Science 2008) CaM-kinase II regulates many neuronal functions 64 g) G-Protein Linked Receptors in Olfaction: Most of our G-protein linked receptors are olfactory receptors (about 350 in humans, 1000 in mouse), located in olfactory receptor cells in the olfactory epithelium in the upper part of the nasal epithelium. Each olfactory cell possess only one type of olfactory receptor, and each receptor can only detect a limited number of odorants. When odorant is detected, receptor stimulates formation of cAMP, which opens cation channels and causes an action potential to fire. We can detect more than 10,000- 1 trillion (?) odors because most odorants interact with more than one receptor: code created for each odor. Olfactory bulb Receptor cells carrying the same type of receptor send their processes to the same glomerulus, micro- regions of the brain. One of the only neuronal cell types that is actively turned over in your life…there are stem cells that maintain these neurons. 67 Review: G-Protein Couples Receptors: --Mediate many different physiological task --All span membrane 7 times --Activate trimeric G-proteins (act as GEF) --G-proteins regulate adenyl cyclase & PLC-b --G-proteins turned off by RGS proteins (act as GAP) --Second Messengers: cAMP, IP3, DAG, Ca2+, --simulate PKA, PKC, CaM-kinases (S/R kinases) --signal amplification --response tightly regulated --target specificity depends on cell context Hedgehog signaling: --route to nucleus through regulating Ci processing