This document provides an overview of the course "Signal Transduction" taught by Prof. Dr. Volker Haucke. It introduces key topics that will be covered in the course, including nuclear hormone receptors, cell surface receptors, G protein-coupled receptors, tyrosine kinase receptors, and communication via gap junctions and ion channels. Nuclear hormone receptors form a superfamily that shares a common design, including a DNA-binding domain with zinc finger motifs and a ligand-binding domain. Hormone binding induces receptor translocation to the nucleus and transcriptional activation.
This document provides an overview of the course "Signal Transduction" taught by Prof. Dr. Volker Haucke. It introduces key topics that will be covered in the course, including nuclear hormone receptors, cell surface receptors, G protein-coupled receptors, tyrosine kinase receptors, and communication via gap junctions and ion channels. Nuclear hormone receptors form a superfamily that shares a common design, including a DNA-binding domain with zinc finger motifs and a ligand-binding domain. Hormone binding induces receptor translocation to the nucleus and transcriptional activation.
This document provides an overview of the course "Signal Transduction" taught by Prof. Dr. Volker Haucke. It introduces key topics that will be covered in the course, including nuclear hormone receptors, cell surface receptors, G protein-coupled receptors, tyrosine kinase receptors, and communication via gap junctions and ion channels. Nuclear hormone receptors form a superfamily that shares a common design, including a DNA-binding domain with zinc finger motifs and a ligand-binding domain. Hormone binding induces receptor translocation to the nucleus and transcriptional activation.
Institut fr Chemie-Biochemie Takustrasse 6 Tel. 030-8385-6920 (secretary) 030-8385-6922 (direct) e-mail: vhaucke@chemie.fu-berlin.de http://userpage.chemie.fu-berlin.de/biochemie/aghaucke/teaching.html Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13; In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13,14 (cell surface signaling) 1. Overview over the molecular mechanisms of signal transduction 2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.) 3. Cell surface receptors & intracellular signaling molecules 4. G protein-coupled receptors 5. Tyrosine kinase & kinase associated receptors (Ras-MAPK pathway, JAK-STAT pathway) 6. Communication by gap junctions and ion channels Signal Transduction Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13; In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13, 14 (cell surface signaling) 1. Overview over the molecular mechanisms of signal transduction 2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.) 3. Cell surface receptors & intracellular signaling molecules 4. G protein-coupled receptors 5. Tyrosine kinase & kinase associated receptors (Ras-MAPK pathway, JAK-STAT pathway) 6. Communication by gap junctions and ion channels Signal Transduction Signals in animal cells a. autocrine (acting on itself) b. paracrine (acting on near neighbour) c. endocrine (carried via blood to distant target cell) Signals in animal cells a. autocrine (acting on itself) b. paracrine (acting on near neighbour) c. endocrine (carried via blood to distant target cell) Characteristics of signal transduction events Characteristics of signal transduction events Characteristics of signal transduction events example: walking from bright sunlight into a dark room Characteristics of signal transduction events example: walking from bright sunlight into a dark room -high affinity of receptors for their ligand (K d very low) -cooperativity in ligand-receptor interaction -amplification of the signal by enzyme cascades Sensitivity in signal transduction General features of signal transduction Step 1: Signal/ ligand interacts with receptor Step 2: Activated receptor interacts with cellular machinery (second signal or change in activity of cellular protein) Step 3: Change in (metabolic) activity of the target cell Step 4: Signal termination (cell returns to prestimulus state) General types of signal transducers General types of signal transducers Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13; In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13, 14 (cell surface signaling) 1. Overview over the molecular mechanisms of signal transduction 2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.) 3. Cell surface receptors & intracellular signaling molecules 4. G protein-coupled receptors 5. Tyrosine kinase & kinase associated receptors (Ras-MAPK pathway, JAK-STAT pathway) 6. Communication by gap junctions and ion channels Signal Transduction Lipid-soluble hormones that bind to nuclear receptors Steroid hormones example derived from Cholesterol (carbohydrate metabolism) (synthesized in: several endocrine tissues incl. adrenal cortex (cortisol), ovaries (estrogens) & testes (testosterone) Lipid-soluble hormones that bind to nuclear receptors Steroid hormones example Retinoids derived from Cholesterol Vitamin A (retinol) (carbohydrate metabolism) (cell differentiation, growth, survival) (synthesized in: several endocrine tissues incl. adrenal cortex (cortisol), ovaries (estrogens) & testes (testosterone) (retinol synthesized in liver from !-carotene; RA synthesized in many tissues) Lipid-soluble hormones that bind to nuclear receptors Steroid hormones example Retinoids Thyroid hormones derived from Cholesterol Vitamin A (retinol) Tyrosine (via iodination & proteolysis of thyroglobulin) (carbohydrate metabolism) (cell differentiation, growth, survival) (stimulates energy yielding metabolism in liver & muscle) (synthesized in: several endocrine tissues incl. adrenal cortex (cortisol), ovaries (estrogens) & testes (testosterone) (synthesized in the thyroid gland) (T 4 ) (retinol synthesized in liver from !-carotene; RA synthesized in many tissues) Nuclear hormone receptors form a superfamily that displays a common design Characteristics: -variable region-C 4 zinc finger DNA binding domain; ligand binding domain -ligand binding domain: contains hormone-dependent activation domain (transcr. activation); may function as repression domain in absence of ligand Nuclear hormone receptors contain a repeat of the C 4 zinc finger DNA binding motif Characteristics: -zinc ions serve to stabilize loop structure and orientation of helices -interaction between DNA & individual zinc finger is weak; usually requires several Zn 2+ fingers -nuclear hormone receptors bind to DNA usually as homo- or heterodimers Nuclear hormone receptors contain a repeat of the C 4 zinc finger DNA binding motif Characteristics: -zinc ions serve to stabilize loop structure and orientation of helices -interaction between DNA & individual zinc finger is weak; usually requires several Zn 2+ fingers -nuclear hormone receptors bind to DNA usually as homo- or heterodimers Hormone response elements (HRE) contain inverted or direct repeats glucocorticoid receptor (GRE) estrogen receptor (ERE) vitamin D 3 receptor (VDRE) thyroid hormone receptor (TRE) retinoic acid receptor (RARE) inverted repeat recognition: by symmetrical homodimeric hormone receptors direct repeat recognition: by heterodimers with common RXR receptor monomer Hormone-dependent gene activation involves translocation of homodimeric nuclear receptors into the cell nucleus 1. hormone diffusion through the plasma membrane 2. binding to receptor (conformational change) & dissociation of inhibitor proteins in the cytoplasm 3. translocation of hormone-receptor complex into the cell nucleus 4. DNA binding & transcriptional activation Hormone-dependent gene activation involves translocation of homodimeric nuclear receptors into the cell nucleus 1. hormone diffusion through the plasma membrane 2. binding to receptor (conformational change) & dissociation of inhibitor proteins in the cytoplasm 3. translocation of hormone-receptor complex into the cell nucleus 4. DNA binding & transcriptional activation Hormone-dependent gene activation involves translocation of homodimeric nuclear receptors into the cell nucleus 1. hormone diffusion through the plasma membrane 2. binding to receptor (conformational change) & dissociation of inhibitor proteins in the cytoplasm 3. translocation of hormone-receptor complex into the cell nucleus 4. DNA binding & transcriptional activation Hormone-dependent gene activation involves translocation of homodimeric nuclear receptors into the cell nucleus + hormone - hormone 1. hormone diffusion through the plasma membrane 2. binding to receptor (conformational change) & dissociation of inhibitor proteins in the cytoplasm 3. translocation of hormone-receptor complex into the cell nucleus 4. DNA binding & transcriptional activation Hormone-dependent gene activation involves translocation of homodimeric nuclear receptors into the cell nucleus 1. hormone diffusion through the plasma membrane 2. binding to receptor (conformational change) & dissociation of inhibitor proteins in the cytoplasm 3. translocation of hormone-receptor complex into the cell nucleus 4. DNA binding & transcriptional activation Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13; In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13, 14 (cell surface signaling) 1. Overview over the molecular mechanisms of signal transduction 2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.) 3. Cell surface receptors & intracellular signaling molecules 4. G protein-coupled receptors 5. Tyrosine kinase & kinase associated receptors (Ras-MAPK pathway, JAK-STAT pathway) 6. Communication by gap junctions and ion channels Signal Transduction Overview of the major classes of cell surface signaling receptors Receptor(s) Ligand(s) Quantitative determination of ligand binding to receptors Quantitative determination of ligand binding to receptors Quantitative determination of ligand binding to receptors bound free [RL] [L] = = 1 K d ([R T ] - [RL]) bound total [RL] [R T ] = = 1 1 + K d / [L] (=R T ) Scatchard Plot Overview of the major classes of cell surface signaling receptors Receptor Second messenger Response (i.e. transcription) Common second messenger as intracellular signaling molecules Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13; In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13, 14 (cell surface signaling) 1. Overview over the molecular mechanisms of signal transduction 2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.) 3. Cell surface receptors & intracellular signaling molecules 4. G protein-coupled receptors 5. Tyrosine kinase & kinase associated receptors (Ras-MAPK pathway, JAK-STAT pathway) 6. Communication by gap junctions and ion channels Signal Transduction Overview of the major classes of cell surface signaling receptors Receptor Second messenger Response (i.e. transcription) General structure of G protein-coupled receptors features: 7 transmembrane "-helices (H1,..,H7) N-term. exoplasmic; C-term. cytosolic C3/ C4 loop as interacting sites for heterotrimeric G proteins G protein-coupled receptors (GPCRs) may signal via different effector pathways including adenylyl cyclase Ligand induced activation of signal-transducing G proteins associated with GPCRs Ligand induced activation of signal-transducing G proteins associated with GPCRs Ligand induced activation of signal-transducing G proteins associated with GPCRs Ligand induced activation of signal-transducing G proteins associated with GPCRs Ligand induced activation of signal-transducing G proteins associated with GPCRs Ligand induced activation of signal-transducing G proteins associated with GPCRs Ligand induced activation of signal-transducing G proteins associated with GPCRs (i.e. adenylyl cyclase [AC]) Ligand induced activation of signal-transducing G proteins associated with GPCRs G protein-coupled receptors (GPCRs) may signal via different effector pathways including adenylyl cyclase Signal cascade involving G protein-coupled receptors (GPCRs) & adenylyl cyclase Epinephrine signals via different types of G protein coupled adrenergic receptors Adrenaline= Epinephrine -released from adrenal gland -regulates energy metabolism in muscle, liver and adipose tissue (i.e. glycogen and fat breakdown) - antagonizes insulin action -four types of receptors: "1, "2, !1, !2; !-receptors found in muscle, liver, and adipose tissue (also used as neurotransmitter in adrenergic neurons) Common second messenger as intracellular signaling molecules Transduction of the epinephrine signal: the !-adrenergic pathway Transduction of the epinephrine signal: the !-adrenergic pathway Adenylyl cyclase (AC): -integral plasma membrane protein -active site on cytoplasmic side Interaction of (active) G s" with adenylyl cyclase AC catalyses the synthesis of cAMP from ATP enzymatic activity stimulated by active G sa Transduction of the epinephrine signal: the !-adrenergic pathway cAMP activates protein kinase A (PKA) Epinephrine signals breakdown of glycogen via !-adrenergic receptor activation Signal amplification in the epinephrine cascade Termination of the cAMP signal Signal termination (1): cAMP hydrolysis via cyclic nucleotide phosphodiesterase (PDE) Caffeine & theophylline: inhibit PDE, increase cAMP half-life, and potentiate AC-mediated effects Signal termination (2): phosphorylation-dependent receptor desensitization (!ARK is a member of the G protein-coupled receptor kinases (GRKs)) G protein-coupled receptors (GPCRs) may signal via different effector pathways including adenylyl cyclase " 1 # and ! 2 -adrenergic receptors are coupled to different G proteins liver, adipose tissue i.e. cardiomyocytes regulation of contraction lipolysis, glycogenolysis Expression Effect 1 G protein-coupled receptors (GPCRs) may signal via different effector pathways including phospholipase C Common second messenger as intracellular signaling molecules Ligand induced activation of signal-transducing G proteins associated with GPCRs (i.e. phospholipase C-! [PLC !]) G "o or G "q Common second messenger as intracellular signaling molecules DAG and IP 3 are second messengers derived from phosphatidyl- inositol 4,5-bisphosphate (PIP 2 ) by PLC-mediated hydrolysis Signaling via DAG and IP 3 involves opening of calcium channels in the plasma membrane & the ER as well as PKC activation Calcium and IP 3 mediated signaling events SUMMARY I - Signaling 1. Signaling in animal cells can be autocrine, paracrine or endocrine and are characterized by specificity, amplification, desensitization, and integration. Scatchard analysis can be used to quantitatively describe receptor-ligand interactions. 2. Steroid & thyroid hormones as well as retinoids bind to nuclear hormone receptors that contain related C 4 -zinc finger DNA binding domains. Upon ligand association dimerized receptors activate gene transcription in the cell nucleus by binding to hormone response elements (HREs) consisting of direct or inverted repeats. 3. G protein-coupled receptors activate heterotrimeric (",!,$) GTP- binding (G) proteins. In the case of the !#adrenergic receptor G sa then activates adenylyl cyclase (AC) which generates cAMP as a second messenger, thus amplifying the signal. 4. Calcium and IP 3 are also second messengers acting downstream of G protein coupled and other receptors that are generated by phospholipase C (PLC)-mediated cleavage of the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP 2 ). Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13; In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13, 14 (cell surface signaling) 1. Overview over the molecular mechanisms of signal transduction 2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.) 3. Cell surface receptors & intracellular signaling molecules 4. G protein-coupled receptors 5. Tyrosine kinase & kinase associated receptors (Ras-MAPK pathway, JAK-STAT pathway) 6. Communication by gap junctions and ion channels Signal Transduction Receptor enzymes - receptor tyrosine kinases The Ras-MAPK pathway regulates development of R7 photoreceptors in flies via a receptor tyrosine kinase sevenless mutation: loss of R7 photoreceptors molecular reason: mutated EGF receptor encoded by sev gene (sev - ) The Ras-MAPK pathway is conserved from mammals to flies where it regulates cell proliferation and development Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs) Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs) Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs) Sos (GEF) binding to Ras facilitates GTP binding by inducing release of GDP via a conformational change that results in Ras activation Ras defines a superfamily of related small GTPases involved in signaling and membrane traffic A kinase cascade transmits signals downstream from activated Ras protein to MAP kinase Cytokine receptors & the JAK-STAT pathway Cytokine receptors & the JAK-STAT pathway Cytokines: family of small (about 160 amino acids), secreted proteins that control cell growth & differentiation examples: IL-2: proliferation of T cells in the immune system interferons: secreted in response to viral infections Epo: produced by kidney cells in response to low oxygen; induces red blood cell differentiation from hematopoietic stem cells Erythropoietin regulates hematopoiesis via a cytokine receptor & the JAK-STAT pathway Erythropoietin binds to a dimeric receptor (EpoR) on erythroid progenitor cells (CFU-E) Erythropoietin binds to a dimeric receptor (EpoR) on erythroid progenitor cells (CFU-E) The Epo receptor (EpoR) and JAK are essential for development of erythrocytes fetal liver (day 13 embryo) Epo binding to its receptor (EpoR) triggers the JAK-STAT signaling pathway JAK (=janus kinase): N-term. receptor binding domain C-term. kinase domain (Tyr kinase) Epo binding: (1) induces conformational change in the EpoR (2) autophosphorylation of JA at Tyr of activation lip (approximation) (3) activated JAK Tyr-phosphorylates EpoR on cytoplasmic tail (4) binding of STAT via SH2 domain to P-Tyr; phosphorylation of C-term. Tyr residues in STAT by JAK Epo binding to its receptor (EpoR) triggers the JAK-STAT signaling pathway JAK (=janus kinase): N-term. receptor binding domain C-term. kinase domain (Tyr kinase) Epo binding: (1) induces conformational change in the EpoR (2) autophosphorylation of JA at Tyr of activation lip (approximation) (3) activated JAK Tyr-phosphorylates EpoR on cytoplasmic tail (4) binding of STAT via SH2 domain to P-Tyr; phosphorylation of C-term. Tyr residues in STAT by JAK Epo binding to its receptor (EpoR) triggers the JAK-STAT signaling pathway JAK (=janus kinase): N-term. receptor binding domain C-term. kinase domain (Tyr kinase) Epo binding: (1) induces conformational change in the EpoR (2) autophosphorylation of JA at Tyr of activation lip (approximation) (3) activated JAK Tyr-phosphorylates EpoR on cytoplasmic tail (4) binding of STAT via SH2 domain to P-Tyr; phosphorylation of C-term. Tyr residues in STAT by JAK Epo binding to its receptor (EpoR) triggers the JAK-STAT signaling pathway (5) STAT dimer goes into the nucleus & activates transcription JAK (=janus kinase): N-term. receptor binding domain C-term. kinase domain (Tyr kinase) Epo binding: (1) induces conformational change in the EpoR (2) autophosphorylation of JA at Tyr of activation lip (approximation) (3) activated JAK Tyr-phosphorylates EpoR on cytoplasmic tail (4) binding of STAT via SH2 domain to P-Tyr; phosphorylation of C-term. Tyr residues in STAT by JAK Termination of the JAK-STAT signaling pathway Dephosphorylation of JAK Blocking and JAK degradation Erythropoietin binds to a dimeric receptor (EpoR) on erythroid progenitor cells (CFU-E) Generation of phosphatidylinositol 3-phosphates downstream of RTKs and cytokine receptors PI 3-kinase: -several isoforms; i.e. p85-p110 dimer -recruited to membrane via SH2 domain downstream of phosphotyrosine formation -PI-3 phosphates produced which transduce signals, i.e. via protein kinase B (PKB; a Ser/ Thr protein kinase) Phosphatidylinositol 3-phosphates activate protein kinase B via binding to its PH domain Protein kinase B (PKB; a Ser/ Thr protein kinase) -PH (=pleckstrin homology) domain of PKB binds to inositol 3-phosphates leading to partial activation at the PM -co-activated by PDK1 which also harbors a PH domain Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13; In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13, 14 (cell surface signaling) 1. Overview over the molecular mechanisms of signal transduction 2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.) 3. Cell surface receptors & intracellular signaling molecules 4. G protein-coupled receptors 5. Tyrosine kinase & kinase associated receptors (Ras-MAPK pathway, JAK-STAT pathway) 6. Communication by gap junctions and ion channels Signal Transduction Gap junctions mediate communication by coupling cells Gap junctions are intercytoplasmic junctions that allow diffusion of small molecules and ions Gap junctions -intercytoplasmic junction -transport of ions & small molecules (<1-2 kDa) (i.e second messengers) between adjacent cells; metabolic coupling (i.e. in muscle to create a syncytium) -opening regulated by calcium, pH etc. -composed of connexons built from 12 connexin proteins (transmembrane protein with 4 transmembrane segments); 2 connexon hemi- channels Neuronal signaling via gated ion channels -transmembrane potential of -60mV (membrane is polarized) is generated by active transport -passive ion fluxes include: a. depolarization by: efflux of anions or influx of cations b. hyperpolarization: influx of anions or efflux of cations The transmembrane electrical potential forms the basis for signaling via voltage- and ligand-gated ion channels The transmembrane electrical potential forms the basis for signaling via voltage- and ligand-gated ion channels -ion fluxes depend on electrochemical gradient: %G 0 = RT lnK + nF V m -ions move passively until equilibrium potential E is reached (%G 0 =0) - specific channel opening leads to change of membrane potential towards E for that ion -action potential first opens Na + , and then K + channels -electrical signal moves along the axon until it reaches nerve terminal -Ca 2+ channels open leading to neurotrans- mitter release -neurotransmitter binds to postsynaptic receptor (ligand-gated ion channels) -ion flux on postsynaptic side may elicit an action potential Ion channels are involved in neuronal signaling Ion channels are involved in neuronal signaling -action potential first opens Na + , and then K + channels -electrical signal moves along the axon until it reaches nerve terminal -Ca 2+ channels open leading to neurotrans- mitter release -neurotransmitter binds to postsynaptic receptor (ligand-gated ion channels) -ion flux on postsynaptic side may elicit an action potential Action potentials involve consecutive opening of voltage-sensitive sodium (Na + ) and potassium (K + ) channels -action potential first opens Na + , and then K + channels -electrical signal moves along the axon until it reaches nerve terminal Ligand-gated ion channels in neuronal signaling -action potential opens Na + and K + channels -electrical signal moves along the axon until it reaches nerve terminal -Ca 2+ channels open leading to neurotrans- mitter release -neurotransmitter binds to postsynaptic receptor (ligand-gated ion channels) -ion flux on postsynaptic side may elicit an action potential Membrane depolarization involves opening of ligand-gated ion channels such as the acetylcholine receptor Gating of the nACh receptor, a ligand-gated ion channel Many postsynaptic neurotransmitter receptors are ligand-gated ion channels Glutamate Glycine nicotinic acetylcholine receptor (AChR) Ligand/ Neurotransmitter Neurotransmitter receptor (ligand-gated ion channel) serotonin receptor glutamate receptors (AMPAR, NMDAR) glycine receptor (GlyR) SUMMARY II - Signaling 1. Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) trigger Sos-induced activation of Ras which in turn activates the MAP kinase cascade to regulate transcription of target genes. 2. Cytokine receptors which are not enzymes themselves among others activate the receptor- associated kinase JAK which becomes autophosphorylated, and further activated by STAT. STAT phosphorylated by JAK dimerizes via its SH2 domain, translocates to the nucleus and activates transcription. The Epo-EpoR system regulates hematopoiesis via the JAK-STAT pathway. 3. Many RTKs or cytokine receptors may also signal via recruitment of phosphatidylinositol 3-kinase and subsequent PH-domain-dependent activation of protein kinase B (PKB). 4. Connexins form connexon hemi-channels that assemble into intercytoplasmic gap junctions that allow electrical coupling between muscle cells or exchange of metabolites and signaling molecules. 5. The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel at the postsynaptic membrane. Opening allows cation flux and may elicit an action potential in the postsynaptic cell.