Physiology Lec 6 Cell signaling F24 (1) PDF

Lecture 6 Cell signaling 1 Topics 1. Introduction 2. Ligands 3. Receptors and their signal transduction pathways 4. Signal inactivation 2 1. Introduction 1. Distinguish between direct and indirect cell signaling....

Lecture 6 Cell signaling 1 Topics 1. Introduction 2. Ligands 3. Receptors and their signal transduction pathways 4. Signal inactivation 2 1. Introduction 1. Distinguish between direct and indirect cell signaling. 2. Describe the structure/function of gap junctions. How do they allow for direct signaling? 3. Describe the basic steps in direct cell signaling. 4. Distinguish among these types of signaling (review, lecture 1): autocrine, paracrine, neuronal, hormonal 3 Direct vs indirect cell signaling Direct: two cells physically Indirect: two cells are not connected; signal travels physically connected, so signal directly from cell A to cell B must leave cell A and find cell B A B A B much more common 4 Direct signaling: gap junctions Junctions found at gap between two cells Two connexons, one from each cell, lock in to form a channel that connects the two cells and lets ions and small molecules, including small signaling molecules, to flow from cell A to cell B. Gap junctions found, e.g., in neurons, smooth and cardiac muscle cells, and epithelial cells Talukdar, S., Emdad, L., Das, S. K., & Fisher, P. B. (2021). GAP junctions: multifaceted regulators of neuronal differentiation. Tissue Barriers, 10(1). https://doi.org/10.1080/21688370.2021.1982349 5 Gap junctions: connexons and connexins Each connexon is a hexameric protein made of 6 (hexa = 6) polypeptides. Each polypeptide is called a connexin. About 20 human connexin genes, but many more connexon proteins, in part because each connexon can be homomeric, or heteromeric Gene mutations can cause disease. LadyofHats, Public domain, via Wikimedia Commons Example: X-linked Charcot-Marie-Tooth disease, a demyelinating peripheral neuropathy. Gap junctions between myelin Schwann cell wrapped layers around axon Söhl, G., Maxeiner, S. & Willecke, K. Expression and functions of neuronal gap junctions. Nat Rev Neurosci 6, 191–200 (2005). https://doi.org/10.1038/nrn1627 6 Indirect signaling 1. Cell A releases chemical messenger, aka ligand or signaling molecule, or Cell B regulatory molecule. (Note: Signal is ECF usually ligand, but it could be other, like light for example.) 2. Ligand/messenger binds receptor molecule on cell B. (Note: receptor could be on or in cell.) chemical 3. The signal coming from the outside is transduced (= changed) to a signal on reactions the inside of the cell: signal transduction Ligand 4. Cell B responds to the transduced signal. Original: Yaneeporn Vector: Pixelsquid🎱, CC BY-SA 4.0 , via Wikimedia Commons 7 Types of indirect signaling Review (lecture 1) autocrine (intrinsic regulation): autocrine signaling molecule paracrine (intrinsic regulation): paracrine signaling molecule neuronal (extrinsic regulation): neurotransmitter hormonal (extrinsic regulation): hormone Remember that except for hormones, ligand diffuses a short distance to receptor. 8 Questions 1-4. Multiple choice 1. connexins forming connexons 2. ligand binding to receptor a) direct 3. Channel connecting two cells lets signaling small signaling molecules flow from one cell to another. b) indirect signaling 4. ligand released from one cell 9 2. Ligands 1. For each type of ligands, identify: polar or nonpolar, basic structure, type of signaling it performs (e.g., paracrine) 2. Identify the basic structure of each type of ligand, and how it functions (e.g., as hormone, etc.) 3. What is the most common type of ligand, and how is it typically processed (hint) post-translationally? 4. Compare how polar vs nonpolar ligands are stored and released. 5. Compare how polar and nonpolar hormones are transported in plasma. 6. What are two roles of plasma carrier proteins? 7. What is albumin, and how is it involved in transport of hormones in blood? 8. Describe how the law of mass action facilitates transport of carried hormones into and out of bloodstream. 9. How many receptors for different ligands can a cell have? Can a receptor bind more than one ligand? Can a ligand bind more than one receptor? 10. How do affinity, ligand concentration, and receptor concentration affect the magnitude of the cellular response? 11. Define: agonist, antagonist 10 Ligand structure Ligands can be polar: water-soluble, hydrophilic, lipophobic nonpolar: fat-soluble, hydrophobic, lipophilic Polar ligands amino acids amines (derived from amino acids; have NH2), peptides, proteins: Nonpolar ligands steroids most common eicosanoids type of ligand nonpolar amines 11 Ligands: amino acids act as neurotransmitters in CNS Glutamate Aspartate Glycine GABA GABA 12 Dancojocari, CC BY-SA 3.0 , via Wikimedia Commons Ligands: amines catechol can act as paracrine signal: histamine neurotransmitter: serotonin catecholamines: dopamine, norepinephrine, epinephrine hormone: thyroid hormone all of these polar except thyroid hormone NEUROtiker, CC BY-SA 3.0 , via Wikimedia Commons thyroid hormone serotonin histamine 13 Ligands: peptides, proteins most ligands are proteins, e.g, insulin can act as: paracrine, neurotransmitter, hormone secreted, so go through secretory pathway, where they often undergo proteolysis post-translationally (lecture 3) prepropetide or preprohormone rough ER propetide or prohormone Golgi hormone 14 Ligands: steroids act as hormones all derived from cholesterol (lecture 2) Later, we’ll learn more about: sex steroids (made in reproductive organs) testosterone (an androgen) estradiol (an estrogen) progesterone (a progestogen) corticosteroids (made in adrenal cortex) aldosterone (a mineralocorticoid) cortisol (a glucocorticoid) https://commons.wikimedia.org/wiki/File:Steroidogenesis.svg#/media/File:Steroidogenesis.svg 15 Ligands: eicosanoids remember: eicosanoids are a type of lipid with 20 (eico) C atoms. act as paracrines; found in most cells most derived from arachidonic acid, a 20-carbon fatty acid found in some phospholipids Include prostaglandins leukotrienes thromboxanes involved in, e.g., inflammation arachidonic acid (AA) By User:Edgar181 - Self-made, en:Image:AAnumbering.png, Public Domain, https://commons.wikimedia.org/w/index.php?curid=1445290 By Jfdwolff, whitespace removed by Fvasconcellos, recreated with editable text by Krishnavedala. - w:Image:Eicosanoid_synthesis.png, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=1619077 16 thyroid hormone  Questions 5-9. Multiple choice Ligands: 5. steroids a) polar 6. amino acids b) nonpolar 7. eicosanoids 8. proteins 9. peptides 17  Questions 10-13. Match 10. amino acids 11. eicosanoids Act as: 12. proteins, peptides, amines a) neurotransmitters b) hormones 13. steroids c) paracrine signals d) all of the above 18 Questions 14-16. Match Derived from : 14. steroids a) amino acids 15. amines b) cholesterol c) fatty acid 16. eicosanoids arachidonic acid 19  Questions 17-20. Match Examples of this type of ligand: 17. glutamate, GABA, glycine 18. dopamine, histamine, serotonin a) eicosanoids 19. prostaglandins, leukotrienes, b) amines thromboxanes c) steroids 20. aldosterone, cortisol, progesterone d) amino acids 20 Ligand storage and release Polar (water-soluble) A storage: in vesicles release: via exocytosis Nonpolar (fat-soluble) A storage: hard to store because it’ll freely diffuse across any membrane, including vesicle or cell membrane, so instead, made as needed release: simple diffusion across membrane 21 Hormone transport in blood Remember: hormones are ligands that travel in blood After ligand is released from cell A and enters bloodstream (whether via simple or facilitated diffusion), it may travel in plasma bound to a carrier protein if nonpolar, can’t dissolve in plasma, so bound to carrier polar hormone carrie if polar, dissolves in plasma, but may also bind to r carrier Being carrier-bound also protects hormone from degradation–good, b/c may take a while to reach target cell! nonpolar carrie r Some carriers are specific for a given hormone; hormone others not Albumin: protein abundant in plasma; has several jobs, including that of nonspecific carrier 22 Plasma hormone concentration and law of mass action Hormone (H) + carrier (C ) HC (hormone-carrier complex) is a reversible reaction. As hormone from cell A enters plasma, the net rate is → HC H+C HC As hormone leaves plasma to get to cell B, the net rate is → H + C H+C HC 23 Review (lecture 4): ligand-receptor binding Binding characterized by: specificity competition saturation affinity  Ligand-receptor: specificity, competition one cell often has >1 type of receptor that bind specific ligand(s) to respond to agonist: I activate receptor different types of signals but one receptor may bind >1 ligand. Ex. norepinephrine and epinephrine may bind the alpha adrenergic receptor. and one ligand may bind more than one natural ligand receptor. Ex. norepinephrine can bind both alpha and beta adrenergic receptors many drugs can also serve as ligands BB agonists: serve as ligands that activate receptor antagonists: serve as ligands that antagonist: I block receptor block receptor Ligand-receptor: affinity, saturation All three graphs: percentage of receptors bound to ligand (y-axis) as function of ligand concentration (x-axis) receptor saturation curve double receptor affinity concentration → shift saturation curve up by 2x Increased affinity, increased ligand or receptor concentration → increase in magnitude of response (magnitude: level, “volume” of response) Question 21. Multiple choice Which is true of carrier proteins? a) they transport lipophilic hormones in blood b) they protect ligand from degradation c) they can be specific or nonspecific for the ligand they carry d) all of the above 27 3. Receptors and their signal transduction pathways 1. Compare receptors for polar vs nonpolar ligands. Where are they located and why? And what is their overall mechanism of action? 2. Compare the two types of nuclear receptors. How are they similar? different? 3. Describe the general structure and mechanism of action of the three main types of cell-surface receptors. Which of the three receptor types is the most common? 4. What is the most common type of enzyme-linked receptor? Describe its general structure and signaling mechanism. 5. Describe the general structure/function of the two types of GPCR effectors. 6. How are GPCR ion channel effectors different from ionotropic ligand-gated ion channels? 7. Describe the steps in the cAMP and PI pathways. Identify the second messenger(s) in each, and where signal amplification occurs. 8. The type of receptor dictates how fast a response is, and how long it lasts. How do the four main types of receptors compare in this sense? 28 Receptors for polar vs nonpolar ligands If ligand polar or lipophobic ○ receptor on cell membrane; cell-surface receptor ○ mechanism of action: doesn’t necessarily involve regulation of gene expression If ligand is nonpolar or lipophilic hormone (not eicosanoids; these bind cell-surface receptors) ○ receptor intracellular; nuclear receptor ○ mechanism of action: involves regulation of gene expression Nuclear receptors: types, and mechanism of action There are two main types of nuclear receptors: ○ Steroid receptors bind steroid hormones. Ex. estrogen receptor ○ Non-steroid receptors bind nonsteroid hormones. Ex. thyroid hormone receptor The signaling mechanism is similar for both: ○ a carrier protein carries the lipophilic hormone in the watery plasma of blood. ○ a hormone-receptor complex binds DNA, acting as a transcription factor (lecture 3) to either up- or down-regulate gene expression. 30 Nuclear receptors: homo- or heterodimers For both steroid and nonsteroid nuclear receptors, you need a pair of receptors, a dimer (di = two), to bind the DNA region. In the case of steroid receptors, the dimer is a homodimer (homo = same) because the two receptors are identical. But in the case of non-steroid receptors, the dimer is a heterodimer (hetero = different) because the two receptors are different. 31 Cell-surface receptors: three main types There are others, but focus on: Ligand-gated ion channels Enzyme-linked receptors (most are RTKs: receptor tyrosine kinases) G-protein coupled receptors (GPCRs) Cell-surface receptors: Ligand-gated ion channels aka: ionotropic receptors, or fast ligand-gated ion channels 1. Ligand binds closed channel 2. Channel opens 3. Ions diffuse → usually change in voltage → cellular response Ligand typically neurotransmitter. Bensaccount at en.wikipedia, Public domain, via Wikimedia Commons For other cell-surface receptors, signaling a bit more complex Diagram: summary of major (not all) signal transduction pathways in mammals RTK and GPCRs, but others A “cascade of events” happens before you get to the cellular response Crosstalk among different pathways serves to regulate cellular responses to different signals. By cybertory - This file was derived from: Signal transduction v1.png, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=12081090 Cell-surface receptors: Enzyme-linked receptors Receptor has enzymatic activity Ligand binds, activating enzyme Most are tyrosine kinase receptors (TKRs), which are made up of two subunits or monomers. a. Ligand binds to each monomer b. monomers come together and auto-phosphorylate. c. This activates receptor into Example of TKR: Insulin receptor in, phosphorylating other for example, skeletal muscle cells molecules in cytoplasm → cascade of events → cellular response Cell-surface receptors: G-protein coupled receptors (GPCRs) aka metabotropic receptors most common type of receptor we have ~800 genes for GPCRs: receptors for many neurotransmitters and hormones; for sensing light, smell, taste all look similar: have 7 transmembrane domains called GPCR because receptor activity is coupled to three G protein subunits (α, β, γ) associated with cell membrane on cytoplasmic side, close to receptor α β γ Fred the OysteriThe source code of this SVG is valid. This vector image was created with Adobe Illustrator by v., CC BY-SA 4.0 , via Wikimedia Commons How GPCRs work: general mechanism, 1 of 2 Ligand 1. Ligand binds → receptor changes shape. G-protein coupled 2. A GDP (guanosine receptor diphosphate) bound to α β γ the alpha (α) G protein subunit is exchanged for a GTP. GTP GDP 37 How GPCRs work: general mechanism, 2 of 2 3. The α subunit dissociates from the βγ dimer. 4. The α and/or βγ move along the membrane, 5. come across effector, and α β activate or inhibit it (see next γ GTP slide) → cascade of events → cellular response. 6. The α subunit has slow GTPase activity. This provides enough time for G protein to “talk” to effector. Effectors 7. The GTP hydrolysis causes the three G protein subunits to go back β to “rest” by the receptor. γ 38 G proteins can stimulate or inhibit effector There are many different groups of G proteins, made by combining different subunits. (There are about 20 α, 5 β, 10 γ.) Each type of GPCR is associated with a particular G protein that is either stimulatory (Gs) or inhibitory (Gi). X Y Gs Gi X receptor is associated with G Y receptor associated with G s i proteins, so effector is activated proteins, so effector is inhibited Two types of GPCR effectors ○ ion channels ○ cell membrane enzymes known as amplifier enzymes GPCR effectors: ion channels aka slow ligand-gated ion channels Note: “ligand-gated” by itself typically refers to ionotropic or fast ligand-gated receptors Channel either opens or closes Different from fast or ionotropic ligand-gated ion channels in that ionotropic ○ always open (not close) ○ are both receptor and effector, not just receptor X Y GPCR effectors: amplifier enzymes amplified volume: volume: low high Amplifier enzymes: amplify the ligand’s signal ○ amplify: increase the magnitude of the cellular response ○ magnitude: “magnification”, “amount” “volume” ○ how? by catalyzing the production of second amplifier messengers, who in turn also help amplify the signal. ligand thus also referred to as primary electrical outlet to power messenger amplification Woodford, Chris. (2009/2022) Amplifiers. Retrieved from https://www.explainthatstuff.com/amplifiers.html. [Accessed 08/2024] We’ll focus on two amplifier enzymes and their associated signaling pathways cAMP pathway ○ effector: adenylate cyclase (AC), an amplifier enzyme ○ 1 second messenger: cAMP ○ pathway named after the second messenger phosphatidylinositol (PI) pathway ○ effector: phospholipase C (PLC) ○ 4 second messengers: diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP3) calcium (Ca++) calmodulin ○ pathway named after PLC substrate, phosphatidylinositol 4,5-bisphosphate (PIP2) By cybertory - This file was derived from: Signal transduction v1.png, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=12081090 cAMP pathway 1. Adenylate cyclase (AC) R AC Amplifier enzyme catalyzes ATP → cAMP Gs amplificatio n: each AC generates 1 2. cAMP activates protein 00s cAMPs kinase A (PKA) 3. PKA phosphorylates many Second messenger other proteins, activating or inactivating them… amplificatio n: each 4. can lead to many different activated PK A cellular responses phosphoryla tes 100s of pro teins Note: Here, the G proteins associated with the receptor are stimulatory (Gs) because they activate the effector. PI pathway PLC catalyzes PIP2 → DAG + IP3 , Amplifier where both DAG and IP3 are second enzyme messengers. DAG stays on membrane, IP3 is released into cytoplasm. Second messengers DAG activates protein kinase C (PKC)... → response IP3 binds to and opens IP3 receptor (a Ca++ channel on ER membrane) causing Ca++ that is stored in the ER lumen to be released into the cytoplasm. Ca++, which is normally kept at very low concentrations in the cytoplasm, acts as a second messenger by interacting with proteins.…→ response. One of the proteins Ca++ can activate is calmodulin, another 2nd messenger that can interact with 100s of molecules….→ response Response rate and duration varies with type of receptor and its associated signaling Type of receptor Response rate: does it Response duration: how happen fast or slow? long does effect last? (ionotropic) ligand-gated ion fast short-lived channel GPCR enzyme-linked (TKR) nuclear slow longer-lived  Questions 22-25. Multiple choice Ligand 22. receptor in cytoplasm or nucleus 23. receptor on cell surface a) lipophilic 24. signaling may not involve b) lipophobic regulation of gene expression 25. ligand-receptor complex act as transcription factor 47 Questions 26-28. Multiple choice 26. channel could open or close Ion channel where receptor is: 27. cellular response is fast a) ionotropic but short-lived b) metabotropic 28. receptor is a different molecule from the effector 48  Questions 29-31. Multiple choice 29. receptor is an ion channel 30. receptor has enzymatic activity a) GPCR 31. receptor is neither an ion b) tyrosine kinase receptor channel nor an enzyme but c) ligand-gated ion channel “talks” to an effector ion channel or enzyme 49 Questions 32. Multiple choice Amplifier enzymes a) are one type of GPCR effector b) help amplify the magnitude of the ligand’s signal by catalyzing the generation of second messengers c) are found on the cell membrane d) all of the above 50 Questions 33. Match Step in any GPCR a) Gs protein stimulates or Gi inhibits effector pathway: b) GTP on G protein alpha subunit → GDP + Pi. 33. step 1 34. step 2 c) GDP comes off alpha G protein subunit, and GTP binds in its place. 35. step 3 36. step 4 d) Ligand binds GPCR, causing receptor to change shape 37. step 5 38. step 6 e) G protein alpha subunit dissociates from 39. step 7 beta/gamma subunits f) G protein subunits return to “rest” by the receptor. g) G protein subunits move along the plane of cell membrane 51 Questions 40-44. Match In the cAMP signal transduction pathway: a) effector b) activated by second 40. adenylate cyclase messenger 41. cAMP c) second messenger 42. PKA d) receptor 43. G protein e) activates effector 44. GPCR 52 Questions 45-49. Match In the PI signal transduction pathway: a) effector 45. PLC b) activated by second 46. DAG, IP3, Ca++, calmodulin messenger 47. PKC, ER Ca++ channel, c) second messenger other proteins or peptides d) receptor 48. G protein e) activates effector 49. GPCR 53 Questions 50-53. Match a) lets Ca++ out of ER into In the PI signal cytoplasm transduction pathway: b) activates PKC 50. DAG c) can bind to many different 51. IP3 proteins to effect different 52. Ca++ responses 53. calmodulin d) increasing its cytoplasmic levels from normally very low levels is used for signaling purposes 54 4. Signal inactivation 1. Why is it important that signaling be inactivated once it happens? 2. Identify ways in which signaling can be inactivated at the level of a. ligand b. receptor c. downstream of receptor 55 Signal must be regulated MSG When ligand stops binding receptor, signal G ends. MS MSG If signaling keeps happening, can cause MSG MSG S G cell injury, even death. M MS ○ Example: too much MSG G (monosodium glutamate) in food. Glutamate (E) is the main excitatory BB neurotransmitter in CNS. Too much E B can over-excite neurons to the point of injury, even death: excitotoxicity. Can inactivate at one or more steps in the Stop over-exciting me! pathway, at level of: https://flic.kr/p/ejqNPc ○ ligand ○ receptor ○ downstream of receptor Ligand inactivation can be done in a number of ways, for example: ○ enzyme can degrade ligand that’s in interstitial fluid ○ cell A can reuptake ligand, then inactivate it ○ ligand can simply diffuse away A A B B ligand degraded in interstitial fluid by enzyme ligand reuptaken and inactivated by cell A Hormone inactivation Hormone can go back to blood, then liver or kidney can metabolize it Hormone in interstitial fluid (not bound to carrier) no longer hormone half-life: carrie r protected from degradation hours ○ half-life of hormone bound to carrier: in range of hours ○ half-life of hormone not bound to carrier: in range of hormone half-life: minutes minutes ○ half-life: how long it takes for ½ of ligand to degrade Other ligands can intervene to inactivate For example: in the case of GPCRs: ligands can bind to receptors Gi associated with inhibitory G proteins (Gi) to inactivate signaling by Gs proteins. Receptor inactivation: role of ligand levels low ligand levels → upregulate receptor expression but high ligand concentration → downregulate receptor expression B cell says: Come pink ligand, we could use more of your signaling here! Cell becomes sensitized to signal BB B B cell says: Ok, that’s too many blue ligands. Let’s not over-signal! Cell becomes desensitized to signal, develops tolerance to signal. Receptor inactivation Can be done in a number of ways. For example, the receptor can be: internalized and internalized and not sequestered degraded internalized, degraded but modified, inactivated Inactivation AD PDE downstream of receptor ATP cAMP AMP Can also happen in a number of ways. For example: in cAMP pathway, Gi phosphodiesterase (PDE) catalyzes cAMP→ AMP. No cAMP = no response. PDE in PI pathway, Ca++ is AMP pumped back into the ER via calcium ATPase. No Ca++ = no response. Questions 54-56. Multiple choice 54. degraded in interstitial fluid Ligand being inactivated 55. reuptaken and could be: inactivated by cell A a) hormone (the cell that released b) neurotransmitter the ligand) c) both 56. transported in blood to liver or kidney to be metabolized 63 Questions 57-59. Match 57. Receptor is kept on endosomal membrane. Mechanism of receptor 58. Receptor is degraded in desensitization: lysosome. a) modification without 59. Receptor remains on cell internalization surface but is modified to be b) sequestration unresponsive to signal. c) degradation 64