HSS2305A - 2024 - Lecture 16 PDF

HSS2305: Molecular Mechanisms of Disease Lecture 16 – Signal Transduction: IP3, DAG and PKC Section A00 Spring 2022 Prof. Keir Menzies Today’s Outline Announcements Signal Transduction: IP3, DAG and PKC Cell Signalling Overview Cell...

HSS2305: Molecular Mechanisms of Disease Lecture 16 – Signal Transduction: IP3, DAG and PKC Section A00 Spring 2022 Prof. Keir Menzies Today’s Outline Announcements Signal Transduction: IP3, DAG and PKC Cell Signalling Overview Cell Signalling signal transduction 2 major routes of message transmission 1. Generation of an intracellular second messenger via an effector enzyme Second messengers activate/inactivate target proteins 2. Recruit signaling proteins to their intracellular domains to initiate a protein activated cascade G Protein-Coupled Receptors https://www.youtube.com/watch?v=jmYn1jJZ9BE G Protein-Coupled Receptors G Protein-Coupled Receptors Effectors 2nd Effector messenger Gs → + Adenylyl Cyclase → cAMP Gi → inhibits Adenylyl Cyclase →  cAMP Gq → + PLCβ →  IP3 + DAG (PLC:phospholipase C) G12/13 → + small G proteins * excessive cell proliferation and malignancies https://www.youtube.com/watch?v=3 qR9B2JCT_s Watch until 14min mark for an overview of Gs, Gi and Gq  G Protein-Coupled Receptors Effectors and 2nd messengers - cAMP Protein Kinase A G Protein-Coupled Receptors Effectors and 2nd messengers - cAMP G Protein-Coupled Receptors Regulation of Blood Glucose The response by a liver cell to glucagon or epinephrine. CREB: cAMP response element binding protein G Protein-Coupled Receptors Effectors and 2nd messengers - cAMP CREB = cAMP response element-binding protein Transcription factor Binds to CRE – response element (TGACGTCA) Fraction of PKA translocate to nucleus→ can phosphorylate CREB → p-CREB Increase transcription of cAMP sensitive genes i.e. gluconeogenesis G Protein-Coupled Receptors Regulation of Blood Glucose The response by a liver cell to glucagon or epinephrine. CREB: cAMP response element binding protein G Protein-Coupled Receptors Effectors and 2nd messengers - cAMP The response by a liver cell to glucagon or epinephrine. CREB: cAMP response element binding protein G Protein-Coupled Receptors Light triggered cell signalling  G Protein-Coupled Receptors Light triggered cell signalling Plasma membrane of Rods G Protein-Coupled Receptors Effectors and 2nd messengers - cGMP Gαt (‘transducin’) Receptor: – Rhodopsin (light-sensitive receptor protein found in the rod cells of the retina ) Effector enzyme = cGMP phosphodiesterase (PDE) Second messengers: – Reduction of cGMP  G Protein-Coupled Receptors Effectors and 2nd messengers - cGMP During the dark cGMP-dependent Na+ and Ca2+ ion channels are open Then when the lights come on: photon light activates GPCR “rhodopsin” Activates G-protein –”transducin” Gαt triggers activation of cGMP phosphodiesterase (effector/enzyme) cGMP→ GMP  cytosolic cGMP closes cGMP- dependent Na+ and Ca2+ ion https://youtu.be/JIPE3in2EcQ channels Membrane hyperpolarization https://youtu.be/AuLR0kzfwBU (membrane becomes more Optional video to watch negative) – signal to visual center G Protein-Coupled Receptors Effectors and 2nd messengers - cGMP Bradyopsia (slow vision) → mutations in genes encoding RGS9 RGS9 normally inactivates transducin during light termination allowing cGMP to increase The result is slower  cGMP → Na+ and Ca2+ channels open slower → slower membrane depolarization (more positive) Patients have trouble adjusting to changing light conditions → temporary blindness when first exposed to bright light G Protein-Coupled Receptors Effectors and 2nd messengers - cGMP AC is an effector for Gs PDE is an effector for Gt G Protein-Coupled Receptors 2nd messengers – IP3 & DAG 2nd Effector messenger Gs → + Adenylyl Cyclase → cAMP Gi → - Adenylyl Cyclase →  cAMP Gq → + PLCβ →  IP3 + DAG G12/13 → + small G proteins * excessive cell proliferation and malignancies G Protein-Coupled Receptors 2nd messengers derived from cell membrane Phospholipids in cell membrane can be targeted for modification and or cleavage by a variety of enzymes Phospholipid kinases - phosphorylation Phospholipid phosphatases – dephosphorylation Phospholipases - cleavage G Protein-Coupled Receptors 2nd messengers derived from cell membrane inositol C5 C4 P 2 Phosphatidylinositol Phosphatidylinositol (PI) 4,5-di-phosphate [PIP2] G Protein-Coupled Receptors 2nd messengers derived from cell membrane Phospholipids in cell membrane can be targeted for cleavage by phospholipase enzymes → cleave at specific sites By-products of cleavage can act as second messengers within the cytoplasm  G Protein-Coupled Receptors 2nd messengers derived from cell membrane Phosphatidylinositol (PI) represents the whole molecule below: PI (Phosphatidylinositol) Inositol phosphate (IP) Diacylglycerol (DAG)  G Protein-Coupled Receptors 2nd messengers derived from cell membrane PIP2 Inositol phosphate 3 (IP3) Diacylglycerol **IP3 → 3 P phosphate P (DAG) groups attached to P ring Phospholipase C (PLC): cleaves phosphatidylinositol di-phosphate (PIP2) → IP3 and DAG IP3 and DAG can act as second messengers within the cytoplasm G Protein-Coupled Receptors 2nd messengers – IP3 & DAG Gαq → Effector enzyme = phospholipase C (PLC) which cleaves PIP2 Second messengers: – inositol trisphosphate (IP3) – diacylglycerol (DAG) Receptors: – Alpha 1 adrenergic receptors (P.S. Alpha 2 adrenergic receptors are the type from the last lecture that induce Gi to reduce cAMP and therefore smooth muscle contraction) – Serotonin receptor (5-HT receptor type 2) – Muscarinic receptors 1, 3, 5 – Histamine receptor type 1 – Calcitonin receptor G Protein-Coupled Receptors 2nd messengers – IP3 & DAG Messenger-2 Effector Messenger-1 The generation of second messengers as a result of ligand-induced breakdown of PhosphoInositides (PIP2) in the lipid bilayer https://youtu.be/lsYBeFqEwzk https://youtu.be/larIxw_9ePU (@ 20 sec)  G Protein-Coupled Receptors 2nd messengers – IP3 & DAG 2 Step 1 and 2 → phosphate groups are added to PI by phospholipid kinases to generate PIP2  G Protein-Coupled Receptors 2nd messengers – IP3 & DAG Step 3 → ligand binds to receptor and activates G protein Replacement of GDP→ GTP on Gαq Ligands = vasopressin; thyroid-stimulating hormone (TSH); angiotensin; neurotransmitters like GABA Step 4 → Gαq activates phospholipase C (PLCβ)  G Protein-Coupled Receptors 2nd messengers – IP3 & DAG Step 5 → activated PLCβ cleaves PIP2 into IP3 (into cytosol) and DAG (remains in cell membrane) Step 6 → DAG recruits protein kinase C (PKC) to membrane and activates the enzyme Cellular growth and differentiation Cellular metabolism Cell death  G Protein-Coupled Receptors 2nd messengers – IP3 & DAG Step 7 and 8 → IP3 diffuses into cytosol and binds to receptor of Ca2+ channel on smooth endoplasmic reticulum (SER) Step 9 → Calcium is released into cytosol to help recruit PKC to DAG and can also have many other effects in the cell i.e. : Contraction Release of histamine G Protein-Coupled Receptors PKC signalling DAG → PKC signalling  G Protein-Coupled Receptors PKC signalling PKC = Family of serine/threonine kinases Α,β,γ Conventional: Require DAG + Ca2+ for activation * δ, ε Novel: Require DAG but not Ca2+ activation Atypical: Require neither DAG or Ca2+ PS: conventional or classic PKC isozymes (cPKCs; α, βI, βII, and γ), novel or nonclassic PKC isozymes (nPKCs; δ, ε, η, and θ), and atypical PKC isozymes (aPKCs; ζ, ι, and λ).  G Protein-Coupled Receptors PKC signalling Abnormal PKC signalling in several diseases CVD, stroke, diabetes, asthma, autoimmunity/inflammation, neurological diseases https://youtu.be/EBRszGDkHxo Nat Commun. 2014 Dec 8;5:5685. doi: 10.1038/ncomms6685. G Protein-Coupled Receptors Ca2+ signalling IP3 → Ca2+ signalling G Protein-Coupled Receptors Ca2+ Signalling Ca2+ probably the most widely used intracellular messengers Increase in intracellular Ca2+ triggers -> muscle contraction exocytosis, e.g., release of neurotransmitters at synapses secretion of hormones like insulin activation of T cells and B cells adhesion of cells to the extracellular matrix apoptosis events mediated by Protein Kinase C (PKC)  G Protein-Coupled Receptors Ca2+ Signalling Cytosolic concentration of Ca2+ is maintained at an extremely low level (about 0.1 mM) Critical for signaling → optimize ‘signal:noise’ ratio Two major mechanisms for maintaining low calcium levels: 1. active transport of Ca2+ from cytosol out of the cell 2. Sequestration of Ca2+ in endoplasmic reticulum  G Protein-Coupled Receptors Ca2+ Signalling Cytosolic concentration of Ca2+ is maintained at an extremely low level (about 0.1 mM) Critical for signaling → optimize ‘signal:noise’ ratio Increases can occur when: Ca2+ enters the cell via voltage-activated Ca2+ channels → depends on the membrane potential  G Protein-Coupled Receptors Ca2+ Signalling Cytosolic concentration of Ca2+ is maintained at an extremely low level (about 0.1 mM) Critical for signaling → optimize ‘signal:noise’ ratio Calcium Induced Calcium Release (CICR) Ryanodine calcium channels and IP3 receptors on the SER respond to calcium to release more calcium (up to 1mM).  G Protein-Coupled Receptors Ca2+ Signalling Cytosolic concentration of Ca2+ is maintained at an extremely low level (about 0.1 mM) Critical for signaling → optimize ‘signal:noise’ ratio IP3Rs (IP3 receptors) and ryanodine receptors: Activation Mechanism: RyRs: Activated by direct binding of Ca²⁺ (CICR). IP₃Rs: Activated by IP₃ binding; Ca²⁺ modulates sensitivity. Function: RyRs: Facilitate rapid and substantial Ca²⁺ release, essential for processes like muscle contraction. IP₃Rs: Mediate Ca²⁺ release in response to extracellular signals, influencing various cellular functions. G Protein-Coupled Receptors Ca2+ Signalling Calcium signaling and fertilization 40-500 million sperm in ejaculate 200-300 make it to oviduct 20-30 make it to oocyte What prevents more than 1 sperm from fertilizing an egg? Polyspermy → lethal Sperm binding receptor activates GPCR → IP3- mediated calcium release (see below)– leads to a wave of calcium that is likely dependent on CICR Cortical Reaction fusion of egg and sperm induces a wave of Ca2+ influx This wave of calcium stimulates calcium granules to release their contents outside the egg → prevent polyspermy by inactivating sperm-binding receptors G Protein-Coupled Receptors Ca2+ Signalling Calcium signaling and fertilization Next Lecture Signal Transduction – RTKs and MAPK (Ch. 15)