Molecular Signaling within Neurons (NESC/PSYO/PHY2570 Lecture 1) PDF

NESC / PSYO / PHYL 2570 Module 2 - Lecture 1 Molecular signaling within neurons Dr. Stefan Krueger Dept of Physiology & Biophysics 1. Molecular signaling within neurons 1.1. General concepts 1.2. Receptors: Types 1.3. G proteins and their t...

NESC / PSYO / PHYL 2570 Module 2 - Lecture 1 Molecular signaling within neurons Dr. Stefan Krueger Dept of Physiology & Biophysics 1. Molecular signaling within neurons 1.1. General concepts 1.2. Receptors: Types 1.3. G proteins and their targets 1.4. Second messengers 1.5. Regulation of gene expression 1.6. Examples of neuronal signal transduction  1.1. General concepts 1.1.1. What are essential components of intercellular communication? 1.1.2. What forms of intercellular communication can be distinguished? 1.1.3. What types of signals mediating intercellular communication can be distinguished? 1.1.4. What are the organizing principles of intracellular signal transduction pathways? 1.1.5. How are protein targets of intracellular signal transduction pathways regulated?  1.1.1. Essential components of intercellular communication Essential molecular signaling components: 4  1.1.2. Forms of intercellular communication Signaling between neurons and non-neuronal cells can be: A. Synaptic B. Paracrine C. Endocrine 5  1.1.3. Types of signals mediating intercellular communication Signalling molecules can be: A.Cell-impermeant B. Cell-permeant C.Cell-associated 6  1.1.4. Principles of intracellular signal transduction pathways (1) Intracellular pathway transduces signal from activated receptor to target 7  1.1.4. Principles of intracellular signal transduction pathways (2) Signal transduction pathways ‣ have multi-layered, hierarchical structure ‣ amplify initial signal ‣ often regulate divergent targets ‣ tightly controlled due to constitutive and regulated feedback mechanisms ‣ often desensitize during continued presence of signal 8  1.1.5. Mechanisms of target protein regulation ‣ Protein targets of signaling cascades often phosphorylated on serine, threonine, or tyrosine residues by serine/threonine kinases and tyrosine kinases ‣ Phosphorylation leads to changes in protein structure or ability of protein to bind other proteins => changes in function (e.g., enzyme with more or less activity) ‣ Dephosphorylation by protein phosphatases reverses change 9  1.2. Receptors 1.2.1. Ionotropic receptors: Alternate terms, activation, effects of activation, structure 1.2.2. Metabotropic receptors: Mechanism and effects of activation, structure 1.2.3. Enzyme-linked receptors: Mechanism and effects of activation 1.2.4. Intracellular receptors: Mechanism and effects of activation  1.2.1. Ionotropic receptors (1) = Ligand-gated ion channels ‣ Binding of ligand (= signal) causes opening of ion channel ‣ Channel is ion-selective ‣ Diffusion of ions into/out of cytoplasm elicits change in membrane potential ‣ If ion channel permeable to calcium, calcium signaling initiated 11 1.2.1. Ionotropic receptors (2): Structure Structure of ionotropic receptors ‣ 4-5 subunits grouped around central pore ‣ Charged amino acid residues at pore entrance form ion selectivity filter ‣ Gate in pore center, opens with ligand binding; can close while ligand bound: Desensitization 12  1.2.2. Metabotropic receptors (1) = G protein-coupled receptors (GPCRs) ‣ Bind heterotrimeric G proteins ‣ Binding of ligand (= signal) causes activation of G protein ‣ G proteins regulate enzymes, ion channels 13  1.2.2. Metabotropic receptors (2): Structure ‣ GPCRs share common structure, span membrane 7 times: Seven-transmembrane receptors ‣ Ligand binding leads to conformational change in receptor that leads to activation of associated G protein 14  1.2.3. Enzyme-linked receptors (1) ‣ Enzyme-linked receptors have intracellular domain that has enzymatic activity: - receptor tyrosine kinases - serine/threonine (S/T) kinases - tyrosine and S/T phosphatases - guanylyl cyclases etc. ‣ Signal binding to extra- cellular domain activates enzyme activity 15  1.2.3. Enzyme-linked receptors (2): Example receptor tyrosine kinases Activation of receptor tyrosine kinases involves: 1) Ligand binding 2) Receptor dimerizes 3) Autophosphorylation 4) Binding of effectors, phosphorylation of other proteins, … 16  1.2.4. Intracellular receptors (1) ‣ Activated by lipophilic signaling molecules that diffuse across PM ‣ Binding of signaling molecule causes disinhibition of receptor (i.e., by dissociation from inhibitory protein) ‣ Cytosolic receptors translocate to eus ‣ Activated receptors bind co- activator proteins and/or DNA to induce gene transcription 17  1.2.4. Intracellular receptors (2) ‣ Activated by lipophilic signaling molecules that diffuse across PM ‣ Binding of signaling molecule causes disinhibition of receptor (i.e., by dissociation from inhibitory protein) ‣ Cytosolic receptors translocate to cell nucleus ‣ Activated receptors bind co- activator proteins and/or DNA to modulate gene transcription 18  1.3. G proteins and their targets 1.3.1. What are general properties of G proteins? 1.3.2. Which two classes of G proteins exist? 1.3.3. How are heterotrimeric G proteins activated and inactivated? 1.3.4. What are types and targets of heterotrimeric G proteins? 1.3.5. How are small monomeric G proteins activated and inactivated? 1.3.6. What are effectors and functions of small G proteins?  1.3.1. Properties of G proteins ‣ G proteins = proteins that are able to bind and hydrolyze GTP ‣ Regulate effectors (enzymes or ion channels) ‣ G proteins interact with effectors when GTP-bound ‣ G proteins inactive in their GDP- bound form 20  1.3.2. Types of G proteins Two classes of G proteins: ‣ Heterotrimeric G proteins: Composed of three distinct subunits (α, β, γ); often activated by metabotropic receptors ‣ Small monomeric G proteins: Single polypeptide; activation by receptor tyrosine kinases and other mechanisms 21  1.3.3. Activation and inactivation of heterotrimeric G proteins 1) Ligand binds to GPCR 2) GPCR promotes exchange of GDP for GTP in G protein 3) G protein α subunit and βγ subunit dissociate, dissipate from receptor 4) Both α and βγ subunit can interact with effectors (enzymes, ion channels) 5) Facilitation of GTP hydrolysis by GTPase activating protein (GAP), subunits re-associate with GPCR 22  1.3.4.Types and targets of heterotrimeric G proteins G protein effector second messenger activates adenylate Gs cAMP↑ cyclase inhibits adenylate Gi cAMP↓ cyclase activates diacylglycerol ↑ Gq phospholipase C IP3 ↑ activates cGMP Gt (transducin) cGMP↓ phosphodiesterase 23  1.3.5. Activation of small monomeric G proteins 1) Guanine nucleotide exchange factor (GEF) facilitates replacement of GDP by GTP, activating G protein 2) GTPase activating protein (GAP) facilitates hydrolysis of GTP, inactivating G protein 3) GEFs can be activated by variety of signals including activated receptor tyrosine kinases 4) GAPs can also be regulated by upstream signalling 24  1.3.6.Targets of monomeric G proteins (1) Examples: effector / monomeric G function downstream protein family pathway cell proliferation, MAP kinase ras differentiation, pathway survival rho actin dynamics ROCK kinase membrane rab various trafficking 25 1.3.6.Targets of monomeric G proteins (2) MAP kinase pathway MAPK = MAP kinase MAPKK = MAP kinase kinase MAPKKK = MAP kinase kinase kinase (a cascade of Ser/Thr kinases) 26  1.4. Second messengers & targets 1.4.1. cAMP : Generation, effectors, degradation Function and regulation of protein kinase A 1.4.2. cGMP : Generation, effectors, degradation, function 1.4.3. IP3 and DAG : – Generation – Effectors 1.4.4. Calcium : – Regulation of basal calcium concentration – Sources of calcium transients – Effectors 1.4.5. Comparison of second messengers and their effectors  1.4.1. cAMP and effectors (I) ‣ cAMP= cyclic adenosine monophosphate ‣ Generated by adenylyl cyclase (activated by Gs, inhibited by Gi proteins) ‣ Activates protein kinase A (PKA) ‣ Also binds to and modulate conductance of cyclic nucleotide-gated ion channels ‣ cAMP degraded by phosphodiesterases 28  1.4.1. cAMP and effectors (2): Protein kinase A Protein kinase A (PKA): ‣ Serine/threonine kinase, phosphorylates proteins involved in synaptic transmission, glucose and lipid metabolism ‣ Two catalytic, two regulatory subunits ‣ cAMP binds to regulatory subunits, relieving inhibition of catalytic subunits 29  1.4.2. cGMP ‣ cGMP = cyclic guanosine monophospate ‣ Generated by guanylyl cyclase ‣ activates protein kinase G (PKG) ‣ bind to and modulates conductance of cyclic nucleotide- gated ion channels ‣ cGMP degraded by GT protein- activated phosphodiesterase ‣ cGMP important second messenger in photoreceptors 30  1.4.3. IP3 and diacylglycerol (1) ‣ Phosphatidylinositol bisphosphate (PIP2) is a phospholipid in the PM ‣ Cleavage by phospholipase C (PLC) yields diacylglycerol (DAG, membrane- associated second messenger) and inositol trisphosphate (IP3, second messenger in cytosol) ‣ Multiple PLC isoforms. Major isoform activated by Gq proteins. 31  1.4.3. IP3 and diacylglycerol (2) ‣ IP3 binds to and activates IP3 receptors, ligand-gated calcium channels in the ER membrane. ‣ Calcium released from ER stores is “third messenger”, initiates Ca2+- dependent signalling (see below) ‣ DAG (and calcium) activates protein kinase C (PKC), a serine/threonine kinase, other proteins (e.g. Munc13) 32  1.4.4. Calcium (1): Maintenance of basal 2+ cytoplasmic Ca concentration Basal Ca2+ concentration in cyto- plasm very low (≤100 nM) due to: ‣ Extrusion by plasma membrane calcium ATPase (PMCA) ‣ Uptake into ER by sarco/endoplasmic reticulum calcium ATPase (SERCA) ‣ Buffering by Ca2+-binding proteins (e.g. calbindin) ‣ Mitochondrial calcium uptake 33 1.4.4. Calcium (2): Sources of Ca2+ signals Transient, spacially restricted calcium signal through opening of: ‣ Voltage-gated calcium channels ‣ Ligand-gated calcium channels ‣ IP3 Receptors (from ER, IP3- gated) ‣ Ryanodine receptors (from ER, Ca2+-gated: Calcium-induced calcium release) 34  1.4.4. Calcium (3): Ca2+ effectors Transient, local calcium elevations activate Ca2+-binding effectors: ‣ Ca2+ activates Calmodulin (CaM) ‣ Ca2+/CaM modulates kinases, voltage-gated Ca2+ channels, … ‣ Modulation of synaptic transmission by Ca2+/calmodulin-dependent kinase II (CaMKII), a serine/threonine kinase 35  1.4.4. Calcium (4): Locally restricted signalling ‣ Due to efficient calcium buffering, extrusion: Calcium signalling often local rather than cell- wide ‣ Example: Compartmentalization of calcium signals in dendritic spines 36 1.4.5. Summary: Second messengers and their effectors ‣ Extracellular signal (e.g., neurotrans- mitter) can often elicit activation of different signaling cascades in diffe- rent target cells ‣ GPCR type deter- mines signaling cascade 37  1.5. Regulation of gene expression 1.5.1. What are principles of gene regulation by signal transduction pathways? 1.5.2. What is the cAMP response element binding protein (CREB) and how is it regulated? 1.5.3. What are immediate early genes? How id c-fos regulated and what are effects of its activation?  1.5.1. Regulation of gene expression by signal transduction pathways: Principles ‣ Synthesis of new mRNA and protein regulated by signal transduction pathways ‣ Onset slow (>30 min), long-lasting ‣ Gene transcription requires binding of transcriptional activator proteins to DNA near promoter of target gene ‣ Binding of transcriptional activator allows formation of RNA polymerase complex, transcription of gene 39  1.5.2. CREB signalling ‣ cAMP-responsive element (CREB) needs to be phosphorylated to have transcriptional activity ‣ phosphorylation and activation by PKA, MAPK, and Ca2+/Calmodulin kinase ‣ Activated CREB stimulates transcription of specific genes 40  1.5.3. c-fos signalling ‣ c-fos: transcriptional activator that is present in unstimulated cells at a very low concentration ‣ c-fos is “immediate early gene”: stimulus (e.g. ras/MAPK, cAMP/PKA, CaMK signal) directly elicits transcription of c-fos ‣ synthesized c-fos protein then stimulates transcription of other genes 41  1.6. Examples of neuronal signal transduction 1.6.1. Example of divergent signal transduction: Nerve Growth Factor (NGF) signalling 1.6.2. Example of convergent signal transduction: Regulation of tyrosine hydroxylase activity 1.6.1. Divergent signal transduction: NGF ‣ NGF = Neurotrophic growth factor ‣ TrkA = NGF receptor, a receptor tyrosine kinase ‣ Required by sensory and sympathetic neurons for survival, differentiation, neurite outgrowth ‣ Activation of multiple signaling pathways: PLC, ras/MAPK, PI3K/Akt 43  1.6.2. Convergent signal transduction: Tyrosine hydroxylase ‣ Tyrosine hydroxylase (TH) catalyses first step in synthesis of catecholamine neurotransmitters ‣ Phosphorylation of TH leads to increase in enzyme activity, increased catecholamine synthesis ‣ TH phosphorylation by PKA, PKC, MAP kinase, CaMKII: Convergent signalling 44