HSS2305A - 2024 - Lecture 15 PDF

HSS2305: Molecular Mechanisms of Disease Lecture 15 – Cell Signalling and G-Coupled Receptors Prof. Keir Menzies Cell Signalling Overview- with an old school video! lol http://www.dnalc.org/resources/3d/cellsignals.html http://www.dnalc.org/resources/3d/ce...

HSS2305: Molecular Mechanisms of Disease Lecture 15 – Cell Signalling and G-Coupled Receptors Prof. Keir Menzies Cell Signalling Overview- with an old school video! lol http://www.dnalc.org/resources/3d/cellsignals.html http://www.dnalc.org/resources/3d/cellsignals.html Cell Signalling Overview Cells must respond adequately to external stimuli to survive Cells respond to stimuli via cell signaling Some signal molecules (or ligands) enter cells; others bind to cell-surface receptors Receptors respond to ligands to initiate intracellular action Message relayed inside cell via a cascade of downstream protein conformational changes → action Cell Signalling Overview Extracellular messenger molecules: transmit messages between cells autocrine signalling → cell has receptors on its surface that respond to the messenger (a) Cells induce or supress themselves – send and respond to same message paracrine signalling → messenger molecules travel short distances through extracellular space (b) Adjacent cells, regulate each other endocrine signalling → messenger molecules (hormones) reach their target cells through the bloodstream (c) Messenger travels far from site of secretion to reach target tissues/organs/cells Cell Signalling how is a signal transduced: signal transduction Ligand Extracellular messages / signal molecule Ex. steroids, neurotransmitters, glucagon, insulin, growth factors Receptors Proteins on target cells that receive messages Conformational change in receptor relays message into cytoplasmic domain 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 Cell Signalling signal transduction Signalling pathways or signalling cascades each protein alters conformation of the next protein protein conformation usually altered by phosphorylation Kinases (+ Pi; ~500) Phosphatases (- Pi; ~150) Amino acids Serine, threonine, tyrosine* Activate/inactivate an enzyme Target proteins ultimately receive a message to alter cell activities such as: Leading to phosphorylation of a TF→ gene transcription Activity of metabolic enzymes Cell mobility DNA synthesis → replication Cell death Cell Signalling signal transduction Protein phosphorylation can change protein behavior of cells in different ways activate/inactivate an enzyme Increase/decrease protein- protein interactions change subcellular location of a protein trigger protein degradation Phospho patterns can differ between cell types (e.g. 2 types of breast cancer cells, which could lead to treatment decisions) Comparison in frequency of tyrosine phosphorylation (red intensity) in 2 types of breast cancer cells. mail Phosphorylation Is a Central Mechanism for Circadian Control of Metabolism and Physiology Maria S. Robles1, Sean J. Humphrey1, Matthias Mann1, 2, , Cell Metabolism 2016 Cell Signalling Receptors Cell Signalling Receptors Receptor types include: a) a) G-protein coupled receptors (GPCRs) b) Receptor protein-tyrosine kinases (RTKs) c) Ligand gated channels b) d) allow or conduct passage of ion d) Steroid hormone receptors nuclear receptors c) e) Specialized receptors i.e. B-and T-cell receptors - immune response to generate diversity in immune response G Protein-Coupled Receptors https://www.youtube.com/watch?v=Glu_T6DQuLU G Protein-Coupled Receptors G protein-coupled receptors (GPCRs) Largest family of membrane proteins by size and diversity 7 transmembrane helical domain receptor Thousands of different GPCRs Coupled to cytoplasmic G protein 3 subunits (α, ß, γ) Uses GTP to activate “effector” protein and trigger second messengers G Protein-Coupled Receptors Small molecules such as amino acids and their derivatives (e.g. acetylcholine, epinephrine, dopamine). Gases such as NO and CO Steroids (regulate sexual differentiation, pregnancy, carbohydrate metabolism) Eicosanoids, which are lipids derived from fatty acids. Various peptides and proteins G Protein-Coupled Receptors G Protein-Coupled Receptors G Protein-Coupled Receptors Heterotrimeric G protein G Protein-Coupled Receptors signal transduction 1. Ligand binding to the extracellular domain alters receptor conformation  affinity and binding of G protein to receptor 2. Upon binding, Gα conformational change GDP is exchanged for GTP = G protein activation A number of G proteins can become activated 3. GTP-Gα dissociates from Gßγ and binds effector has low affinity for Gßγ subunits 4. GTP-Gα can activate/inhibit an effector protein Adenylyl cyclase → convert ATP to cAMP (2nd messenger) Phospholipase C cGMP phosphodiesterase G Protein-Coupled Receptors signal transduction 1. Ligand binding to the extracellular domain alters receptor conformation  affinity and binding of G protein to receptor 2. Upon binding, Gα conformational change GDP is exchanged for GTP = G protein activation A number of G proteins can become activated 3. GTP-Gα dissociates from Gßγ and binds effector has low affinity for Gßγ subunits 4. GTP-Gα can activate/inhibit an effector protein Adenylyl cyclase → convert ATP to cAMP (2nd messenger) Phospholipase C cGMP phosphodiesterase G Protein-Coupled Receptors signal transduction 1. Ligand binding to the extracellular domain alters receptor conformation  affinity and binding of G protein to receptor 2. Upon binding, Gα conformational change GDP is exchanged for GTP = G protein activation A number of G proteins can become activated 3. GTP-Gα dissociates from Gßγ and binds effector has low affinity for Gßγ subunits 4. GTP-Gα can activate/inhibit an effector protein Adenylyl cyclase → convert ATP to cAMP (2nd messenger) Phospholipase C cGMP phosphodiesterase G Protein-Coupled Receptors signal transduction 1. Ligand binding to the extracellular domain alters receptor conformation  affinity and binding of G protein to receptor 2. Upon binding, Gα conformational change GDP is exchanged for GTP = G protein activation A number of G proteins can become activated 3. GTP-Gα dissociates from Gßγ and binds effector has low affinity for Gßγ subunits 4. GTP-Gα can activate/inhibit an effector protein Adenylyl cyclase → convert ATP to cAMP (2nd messenger) Phospholipase C cGMP phosphodiesterase G Protein-Coupled Receptors signal transduction 5. Gα subunit can turn itself “off” via hydrolysis of GTP → GDP + Pi 6. Conformational change of Gα decreases affinity for effector protein and increases affinity for Gßγ subunits 7. Can be Desensitized: receptor is phosphorylated by G protein- coupled receptor kinase (GRK) 8. Desensitization step 2: Arrestin protein binds to receptor and inhibits binding of G protein Promote internalization of GPCRs Internal signalling Degradation Dephosphorylated and returned to cell membrane (re- sensitized) G Protein-Coupled Receptors signal transduction 5. Gα subunit can turn itself “off” via hydrolysis of GTP → GDP + Pi 6. Conformational change of Gα decreases affinity for effector protein and increases affinity for Gßγ subunits 7. Can be Desensitized: receptor is phosphorylated by G protein- coupled receptor kinase (GRK) 8. Desensitization step 2: Arrestin protein binds to receptor and inhibits binding of G protein Promote internalization of GPCRs Internal signalling Degradation Dephosphorylated and returned to cell membrane (re- sensitized) G Protein-Coupled Receptors signal transduction 5. Gα subunit can turn itself “off” via hydrolysis of GTP → GDP + Pi 6. Conformational change of Gα decreases affinity for effector protein and increases affinity for Gßγ subunits 7. Can be Desensitized: receptor is phosphorylated by G protein- coupled receptor kinase (GRK) 8. Desensitization step 2: Arrestin protein binds to receptor and inhibits binding of G protein Promote internalization of GPCRs Internal signalling Degradation Dephosphorylated and returned to cell membrane (re- sensitized) G Protein-Coupled Receptors signal transduction 5. Gα subunit can turn itself “off” via hydrolysis of GTP → GDP + Pi 6. Conformational change of Gα decreases affinity for effector protein and increases affinity for Gßγ subunits 7. Can be Desensitized: receptor is phosphorylated by G protein- coupled receptor kinase (GRK) 8. Desensitization step 2: Arrestin protein binds to receptor and inhibits binding of G protein Promote internalization of GPCRs Internal signalling Degradation Dephosphorylated and returned to cell membrane (re- sensitized) G Protein-Coupled Receptors signal transduction 5. Gα subunit can turn itself “off” via hydrolysis of GTP → GDP + Pi 6. Conformational change of Gα decreases affinity for effector protein and increases affinity for Gßγ subunits 7. Can be Desensitized: receptor is phosphorylated by G protein- coupled receptor kinase (GRK) 8. Desensitization step 2: Arrestin protein binds to receptor and inhibits binding of G protein Promote internalization of GPCRs Internal signalling Degradation Dephosphorylated and returned to cell membrane (re-sensitized) G Protein-Coupled Receptors signal transduction Strength and duration of signal are determined by rate of GTP hydrolysis by Gα subunit Gα subunit possess weak GTPase activity → slowly hydrolyzes GTP and inactivates themselves Termination of response can be accelerated by Regulators of G protein Signaling (RGSs; not seen in diagram here) Increases rate of GTP hydrolysis G Protein-Coupled Receptors  G Protein-Coupled Receptors Effectors 2nd Effector messenger Gs → + Adenylyl Cyclase → cAMP Gs Gi Gi → inhibit Adenylyl Cyclase →  cAMP https://www.youtube.com/watch?v= qOVkedxDqQo G Protein-Coupled Receptors Effectors 2nd Effector messenger Gs → + Adenylyl Cyclase → cAMP Gi → inhibit Adenylyl Cyclase →  cAMP Gq → + PLCβ →  IP3 + DAG (PLC:phospholipase C) G Protein-Coupled Receptors Effectors 2nd Effector messenger Gs → + Adenylyl Cyclase → cAMP Gi → inhibit Adenylyl Cyclase →  cAMP Gq → + PLCβ →  IP3 + DAG G12/13 → + small G proteins * excessive activation leads to cell proliferation and malignancies G Protein-Coupled Receptors Effectors G Protein-Coupled Receptors second messengers Second messengers → released into the cytoplasm after binding of a ligand Many can be created by one effector enzyme in order to amplify the response to a single extracellular ligand 3 common second messenger systems: cyclic nucleotides cAMP and cGMP Phosphatidylinositol derivatives inositol trisphosphate (IP3) and diacylglycerol (DAG) calcium ions (Ca2+) G Protein-Coupled Receptors second messengers - cAMP cAMP = cyclic adenosine monophosphate Synthesized by effector adenylyl cyclase ATP → cAMP Stimulated by Gs subunit Inhibited by Gi subunit Capable of diffusing to other sites within the cell Can stimulate a variety of cellular activities G Protein-Coupled Receptors second messengers - cAMP cAMP→ Activates protein kinases → phosphorylation of proteins Cytoskeleton Protein synthesis Glycogen breakdown/formation → glucose regulation Fatty acid formation Activation of calcium channels G Protein-Coupled Receptors second messengers - cAMP cAMP→ Activates protein kinases → phosphorylation of proteins Cytoskeleton Protein synthesis Glycogen breakdown/formation → glucose regulation Fatty acid formation Activation of calcium channels G Protein-Coupled Receptors second messengers - cAMP cAMP→ Activates protein kinases → phosphorylation of proteins Cytoskeleton Protein synthesis Glycogen breakdown/formation → glucose regulation Fatty acid formation Activation of calcium channels  G Protein-Coupled Receptors second messengers - cAMP Protein Kinase A is activated by cAMP “cAMP-dependent protein kinase” Holoenzyme a biochemically active compound formed by the combination of an enzyme with a coenzyme Subunits: regulatory subunit dimer (R) catalytic subunit (C) low levels of cAMP → catalytically inactive Represents any high levels of cAMP → cAMP binds number of regulatory subunits, releasing the catalytic subunits enzymes Free catalytic subunits can phosphorylate proteins >100 substrates When phosphorylated cAMP phosphodiesterase (PDE) can break down cAMP → feedback loop  G Protein-Coupled Receptors second messengers - cAMP Protein Kinase A Anchoring Proteins (AKAPs): Function as signalling hubs → scaffold for coordinating protein-protein interactions by sequestering PKA to specific locations around the cell > 50 = PKA = AKAP G Protein-Coupled Receptors second messengers - cAMP  G Protein-Coupled Receptors adrenergic receptors Adrenergic receptors or adrenoceptors A class of G protein-coupled receptors targets of catecholamines Ex. norepinephrine (noradrenaline) and epinephrine (adrenaline) GPCRs for epinephrine 9 different receptors grouped as α- and β-receptors Responsible for fight-or-flight response Different tissues and organs have different adrenergic receptors Yield different internal signaling Ex. ß-Adrenergic receptor on cardiac muscle cell → Gs subunit activated → stimulates cAMP production Muscle Muscle → increased rate and force of contraction relaxation contraction Ex. α-Adrenergic receptor on intestinal smooth muscle cell → Gi subunit activated Beta blockers, also known as beta-adrenergic blocking agents, are medications that reduce your blood pressure. → inhibits cAMP production Beta blockers work by blocking the effects of the hormone → muscle relaxation epinephrine, also known as adrenaline. Beta blockers cause your heart to beat more slowly and with less force, which lowers blood pressure.  G Protein-Coupled Receptors Regulation of Blood Glucose Glucose Regulation cAMP leads to glucose mobilization 1. GPCR (cAMP-mediated) responses: Glucagon (*Endocrine Hormone) Released from pancreas in response to low glucose Activates Glucagon GPCR in Liver Stimulates breakdown of glycogen and release of glucose Acts via GPCR (glucagon receptor) Epinephrine (Endocrine Hormone) Released from adrenal gland – fight or flight Activates Adrenergic GPCR in Liver Stimulates breakdown of glycogen and release of glucose Acts via GPCR (ß-Adrenergic receptor ) 2. RTK (receptor protein-tyrosine kinases) responses: Insulin (Endocrine Hormone) Released from pancreas in response to high glucose Stimulates glucose uptake and storage as glycogen * Acts via receptor protein-tyrosine kinases (RTKs) – we’ll cover this in the upcoming lectures *Endocrine hormones are signaling molecules that regulate the physiology and behavior of multicellular organisms. 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 Regulation of Blood Glucose The response by a liver cell to glucagon or epinephrine. CREB: cAMP response element binding protein G Protein-Coupled Receptors Regulation of Blood Glucose The response by a liver cell to glucagon or Liver epinephrine. CREB: cAMP response element binding protein 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 Regulation of Blood Glucose 2. RTK (receptor protein-tyrosine kinases) responses: Insulin Released from pancreas in response to high glucose Stimulates glucose uptake and storage as glycogen * Acts via receptor protein-tyrosine kinases (RTKs) – we’ll cover this in the upcoming lectures This will be covered later…. To be continued in later lectures G Protein-Coupled Receptors Human disease Bacterial Toxins: G proteins are so vital to normal physiology → that they are ideal targets for pathogens Gs→ Target of cholera toxin from the bacteria Vibrio cholerae Modifies Gs subunit such that it cannot exert GTPase activity prolonged Adenylyl Cyclase activation → excessive cAMP production high levels of cAMP causes a massive loss of salts/H 20 from intestinal epithelia profuse watery diarrhea, dehydration The diarrhea induced by cholera toxin benefits V. cholerae by enhancing transmission of the bacteria (ie contaminating water sources or the environment so it can find new hosts) Diarrhea in cholera results from stimulation of cAMP- mediated intestinal Cl− secretion by cholera toxin G Protein-Coupled Receptors Human disease G Protein-Coupled Receptors Human disease Case Study: 8 Year old female brought to ER for fractured bone history of several bone fractures on X-ray evidence of fibrous dysplasia Normal bone tissue replaced by fibrous bone tissue onset of puberty at 7 (precocious puberty) breast development menstruation at 8 developed several irregular, large patches of dark pigmentation on skin throughout childhood G Protein-Coupled Receptors Human disease McCune-Albright Syndrome: Abnormalities in GNAS1 gene (Chr 20) GNAS1 encodes Gαs Compromised GTPase activity constitutive activation of cAMP driven pathways in the absence of hormone stimulation Resulting pathophysiology: 1. Fibrous dysplasia 2. Endocrine hyper-function Precocious puberty (despite low circulating levels of hormones), hyperthryoidism, gigantism 3. Cutaneous hyperpigmentation  G Protein-Coupled Receptors Human disease McCune-Albright Syndrome: Signalling molecules that rely on GPCRs: MSH - melanocyte-stimulating hormones LH – luteinizing hormone GNRH - Gonadotropin-releasing hormone TSH – thyroid stimulating hormone Activating mutation of Gαs GTP Inappropriate signalling Hydrolysis Results in abnormally high amounts of: Melanin - pigmentation Estrogen (LH increases production of estrogen by ovary) – puberty Growth Hormone - gigantism Thyroid hormones – abnormal bone turnover and hypothyroidism Next Lecture Signal Transduction – IP3, DAG and PKC (Ch. 15)