Cell Signaling I: Signal Transduction & Short-term Cellular Responses PDF

Cell Signaling I: Signal Transduction & Short- term Cellular Responses Reminder of structure of Biomembranes Hydrophobic core: impermeable barrier – Prevents H20 soluble (hydrophilic) solutes across the membrane – Membrane proteins modulate this barrier by mediating transport of spe...

Cell Signaling I: Signal Transduction & Short- term Cellular Responses Reminder of structure of Biomembranes Hydrophobic core: impermeable barrier – Prevents H20 soluble (hydrophilic) solutes across the membrane – Membrane proteins modulate this barrier by mediating transport of specific molecules across Strength of bilayer – from hydrophobic & van der Waals interactions b/w lipid chains  General Principles of Signaling by Cell Surface Receptors Hydrophilic chemical signals that bind to cell-surface receptors activate signal- transduction pathways – Ligand binding to the receptor – Transduction via 2nd messengers, cascade – response(s)  Types of Signals Act outside the organism – Pheromones: a signaling molecule released by an individual that can alter the behavior or gene expression of another individual of the same species Yeast mating-type factors Act within the organism – Extracellular signaling molecules that control metabolic processes within cells growth & differentiation of tissues synthesis and secretion of proteins composition of intracellular and extracellular fluids  Schemes of Intercellular Signaling Endocrine: act on target cells at a long distance from their site of synthesis Paracrine: signaling molecules released by a cell affect target cells in close proximity Note: some molecules can serve as both paracrine & endocrine signals (eg. epinephrine as neurotransmitter & systemic hormone) Autocrine: cells respond to substances that they themselves release Juxtacrine: some membrane- bound proteins act as signals How do Signaling Molecules Work? 1. Change the activity or function of a pre-existing protein. 2. Change the amount of protein in a cell by modifying transcription factors, leading to activation or suppression of gene transcription. Response 1 generally occurs more rapidly. Why? General Steps of Extracellular Signaling 1. Synthesis of signaling molecule 2. Release of signaling molecule 1 3. Transport of signaling molecule to target cell 4. Binding of signal by a receptor leading to its activation 2 5. Initiation of intracellular signal-transduction pathways by the activated receptor 3 6. Specific changes in cellular function, metabolism, or development 7. Removal of the signal 4  Common Properties of Cell-Surface Receptors All receptors are proteins Receptors have high ligand binding specificity – Ability to distinguish closely related substances Ligands, on the other hand, exhibit binding versatility – one type of ligand may bind to different types of receptors to activate different pathways Receptor-ligand complex exhibits effector specificity – a specific cellular response is turned on Interaction between Receptor and Ligand Binding of ligand to receptor is by weak, non-covalent interactions (ionic, hydrophobic, van der Waals) – molecular complementarity b/w interacting surfaces of a receptor and ligand Patches of amino acids in receptors and ligands determine their highly specific mutual interaction  Common Properties of Cell-Surface Receptors Ligand binding can be viewed as a simple reversible reaction: R + L kon RL koff KD (Dissociation constant) = [R][L]/[RL] – KD is the concentration of ligand at which half of its receptors are occupied – measures the affinity of the receptor for the ligand/substrate – The lower the KD value, the higher the affinity of a receptor for its ligand Receptors & Associated Signal-Transduction Proteins may be Localized Some receptors may be uniformly distributed on the cell surface; however, others are localized to particular regions. Clustering may be mediated by adapter domains of particular cytosolic proteins (PDZ, SH3, etc.) – PDZ domain is a common element in several cytosolic proteins that bind to integral plasma membrane proteins Small domain, 90 aa residues, that binds to sequences at the C- terminal of target proteins Example: Proteins containing PDZ domains organize and localize receptors in the plasma membrane of the post-synaptic cell – Most cell surface receptors contain multiple subunits, each of which can bind to a PDZ domain – Many cytosolic proteins contain multiple PDZ domains that participate in multiple protein-protein interactions simultaneously Multiple Protein-protein Interactions: Membrane Protein Clustering PDZ domain binds to certain C-term sequences SH3 domain binds to proline rich sequences A single actin filament can bind many these clusters, so large numbers of plasma membrane proteins can be specifically clustered together Result: many receptors binding same or different ligands are localized to a specific region of the plasma membrane  Highly Conserved Components of Intracellular Signal-transduction Pathways 1. GTP-binding 2. Protein switch proteins kinases 3. Protein 4. 2nd phosphatases messengers  GTPase Switch Proteins Belong to GTPase superfamily Guanine nucleotide-binding proteins – are “on” when GTP bound – are “off” when GDP bound After activation, switch 1 & II segments remove P from GTP, cause inactivation Signal induced conversion of inactive to active state is mediated by GEF (Guanine nt-exchange factor), release of GDP, allowing GTP to bind Classes of GTPase Switch Proteins – Trimeric: directly bind & are activated by cell surface receptors – Monomeric: indirectly linked to receptors via adaptor proteins eg. Ras  Kinases & Phosphatases Protein kinases and phosphatases are employed in virtually all signaling pathways Protein Kinases: enzymes that phosphorylate other proteins – Animals cells contain 2 types: one phosphorylates the -OH group of tyrosine one that phosphorylates the -OH group of serine, threonine, or both Protein Phosphatases: Enzymes that remove phosphate groups (dephosphorylate) from other proteins  Second Messengers Binding of ligands to cell-surface receptors leads to the activation of non-protein intracellular molecules called second messengers that transduce signals cAMP (cyclic AMP): activates protein kinase A cGMP (cyclic GMP): activates protein kinase G DAG (Diacylglycerol): activates protein kinase C IP3 (inositol triphosphate): causes Ca2+ release from ER Ca2+: activates a variety of proteins that cause cellular responses, such as: – Muscle contraction in muscle cells – Release of neurotransmitters (vesicle exocytosis in nerve cells) – Release of hormones (exocytosis in endocrine cells) Common Intracellular Second Messengers  G Protein Coupled Receptor Systems Activation of receptor (GPCR) by its ligand will activate the coupled trimeric G protein which interacts with downstream signal transduction proteins 4 elements: – GPCR – Coupled trimeric G protein switch – Membrane bound effector protein – Feedback regulation & desensitization GPCR have short term effects in cell that quickly modify existing proteins – Eg. Enzymes or ion channels Examples of GPCR family members: – Some hormone & neurotransmitter receptors – Light activated receptors in the eye (rhodopsin) – Odorant receptors in the nose  G-Protein Coupled Receptors (GPCR) N-term in the exoplasmic face C-term in the cytosolic face 7 membrane-spanning regions, H1-H7 4 extracellular segments, E1-E4 4 cytosolic segments, C1-C4 – C3 and C4 domains interact with trimeric G-proteins  Structure of Trimeric G Protein 3 subunits: G, G, and G G and G subunits remain together – referred to as the G subunit G subunit is the GTPase – Switch protein that alternates between on (active) & off (inactive)  Multiple G Proteins in Eukaryotic Genome All effector proteins in GPCR PWs are: - membrane bound ion channels - enzymes the catalyze formation of second messengers. e.g., Adenylyl cyclase Most effector proteins are activated by G-GTP, but some are inhibited In some cells, the G subunit signals to the effector protein The activity of several different effector proteins is controlled by different GPCR-ligand complexes. The different G subunits function similarly, where as the G class show functional differences  Ligand Induced Activation of Effector Proteins through GPCR Stimulation Activation of Modulation of Activity of of GPCR G protein associated effectors protein Examples of Effector proteins: Membrane bound ion channels Enzymes that catalyze formation of 2nd messengers GPCR activate exchange of GTP (for GDP) on  subunit of trimeric G protein General Mechanism of GPCR Activation of Effector Proteins  Some GPCR Regulate Ion Channels Effector proteins: some are Na+ or K+ channels – Binding of ligand to GPCR will result in activation of 2nd messengers subsequently the associated ion channel will open or close leads to changes in membrane potential Examples include: – Some neurotransmitter receptors – Cardiac muscarinic acetylcholine receptors – Odorant receptors in nose – Photoreceptors in eye  Muscarinic Acetylcholine Receptors in Heart Muscle Binding of acetylcholine to muscarinic acetylcholine receptors in cardiac muscles are inhibitory (coupled to a Gi protein) – Slows the rate of heart muscle contraction acetylcholine binding causes receptor to bind to Giα subunit GDP is replaced by GTP Activated Giα subunit dissociates from Gβγ subunit Gβγ subunit binds to K+ channel (effector protein) K+ channel opens K+ flow out of the cell causing hyper-polarization frequency of heart muscle contraction decreases 2 General Types of GPCR that Activate or Inhibit Adenylyl Cyclase β-adrenergic receptors: α-adrenergic receptors: these are coupled to these are coupled to an stimulatory Gs protein inhibitory Gi protein stimulate adenylyl cyclase inhibit adenylyl cyclase increase [cAMP] decrease [cAMP]  Activation of Adenylyl Cyclase Signal binds to receptor, receptor undergoes conformational change, becomes active, activated receptor binds to G subunit G subunit undergoes a conformational change – GDP dissociates & GTP binds, G dissociates from G subunit G binds to effector protein, adenylyl cyclase, thereby activating it Hydrolysis of GTP to GDP within a few seconds inactivates G, it re-associates w/ G – this terminates effector activation (adenylyl cyclase inactivated) Adenylyl Cyclase  Function of Adenylyl Cyclase Adenylyl cyclase catalyzes synthesis of cAMP from ATP cAMP activates Protein Kinase A (PKA) by releasing catalytic subunits PKA activates additional proteins, leads to a cellular response – cAMP-activated PKA mediates various responses in different cells  Epinephrine Hormone that is important in mediating body’s response to stress & heavy exercise when tissues need to produce more energy (ATP) from glucose catabolism Liberation of glucose can be triggered by epinephrine binding to their receptors All epinephrine receptors are GPCRs – Different types of receptors are coupled to different G proteins and stimulate different intracellular signaling pathways  Glycogen Metabolism Glycogen = large glucose polymer – major glucose storage form in animals – synthesized by one set of enzymes, degraded by another Incorporation of glucose Removal of glucose units (Glucose 1-P)  Glycogen Metabolism Glycogen metabolism is regulated by epinephrine, which increases cAMP, and induces activation of PKA Epinephrine binding to β-adrenergic GPCRs in muscle and liver cells leads to increased cAMP production. – In both muscle and liver cells, glycogen is broken down to Glucose-1 P, and converted to Glucose-6 P.. In muscle cells: Glucose-6 P enters glycolysis for production of ATP used to power muscle contraction. In liver cells: the Glucose-6 P is hydrolyzed to glucose which is exported from these cells by a plasma membrane glucose transporter (GLUT2).  Glycogen Metabolism If epinephrine is removed, the level of cAMP drops, PKA is inactivated – Mediated by phosphoprotein phosphatase (PP) which reverses the PKA effects.  Role of PKA in Glycogen Metabolism PKA enhances glycogen metabolism in 2 ways: Directly: PKA inhibits glycogen synthase – PKA phosphorylates and inactivates glycogen synthase – Stops glucose incorporation into building glycogen Indirectly: stimulates glycogen phosphorylase – Stimulates degradation of glycogen by phophorylating and activating glycogen phosphorylase kinase (GPK) GPK then phosphorylates and activates glycogen phosphorylase  Regulation of Glycogen Metabolism by cAMP 1. Direct (3. At high [cAMP] 2. Indirect PKA phosphorylates inhibitor) * * * * * * * = active * * *  Signal Amplification activation of higher numbers of molecules downstream from receptor – especially common with cell surface receptors – signaling molecules involving 2nd messengers and kinase cascades amplify an external signal tremendously Amplification of 104  GPCRs that Activate Phospholipase C Involve 2nd messengers that have inositol, which can be reversibly phosphorylated Example: PIP2= PI 4,5-bisphosphate (membrane-bound) – binds many cytosolic proteins to the plasma membrane PLC = phospholipase C – Plasma membrane associated enzyme – Cleaves PIP2 to generate two important 2nd messengers: DAG: 1,2-diacylglycerol, lipophilic molecule, remains in membrane IP3: inositol 1,4,5- triphosphate which diffuses in the cytoplasm  IP3/DAG Pathway & Release of ER Ca2+ Ligand binding to GPCR causes activation of PLC IP3 diffuses thru cytosol, binds IP3- gated Ca2+ channel on smooth ER Fig 15-34 Ca2+ released, causes PKC recruitment to plasma membrane, where it interacts with DAG and is activated.  Activation of PKC PKC phosphorylates and activates cellular enzymes & receptors This leads to various cellular response in different cells, playing key roles in different aspects of cellular growth & metabolism – eg. PKC regulates glycogen metabolism by phosphorylating & inhibiting glycogen synthase. – In many cells, PKC phosphorylates transcription factors that are localized in the cytosol, triggering their movement into the nucleus, where they activate genes necessary for cell division  Ca2+/Calmodulin Complex Release of Ca2+ into the cytosol from IP3-mediated processes can lead to a variety of cellular responses – Localized increases in cytosolic Ca2+ in specific cell types are critical to its function as a 2nd messenger – eg. Acetylcholine stimulation of GPCR in secretory pancreas cells causes IP3-mediated Ca2+ rise, triggering fusion of secretory vesicles with plasma membrane & content release Calmodulin: a small cytosolic Ca2+ binding protein functioning as a multipurpose switch protein mediating multiple Ca2+ cellular effects – binds to 4 Ca2+ ions to form a complex that interacts w/ & modulates the activity of many other proteins & enzymes  Various Processes are Activated by the Ca2+/Calmodulin Complex 1. Activates protein kinases, which phosphorylate TS factors, which modifies their activity and regulate gene expression 2. Plays a key role in controlling diameter of blood vessels  Various Processes are Activated by the Ca2+/Calmodulin Complex 3. Activates phosphatases that dephosphorylate TS factors – Example: NFAT= nuclear factor of activated T cells Essential TS factor enhanced by Ca2+ ions In unstimulated cells, phosphorylated NFAT in cytosol After stimulation, cytosolic [Ca2+] increased, Ca2+/Calmodulin complex binds to and activates calcineurin (a phosphatase) Activated calcineurin dephosphorylates NFAT, exposing NLS NFAT moves into the nucleus, stimulates expression of genes essential for T cell activation NFAT SUMMARY – steps summary SUMMARY