Cell Signaling Handouts PDF

Cell Signaling Nukhet Aykin-Burns, PhD Department of Pharmaceutical Sciences BioMed II Room: 440-2 [email protected]  Learning Objectives: At the end of this lecture, the students should be able to: Understand the concepts of signaling molecule, recep...

Cell Signaling Nukhet Aykin-Burns, PhD Department of Pharmaceutical Sciences BioMed II Room: 440-2 [email protected]  Learning Objectives: At the end of this lecture, the students should be able to: Understand the concepts of signaling molecule, receptor, intracellular transducer, effector protein, biosignaling cascade (signal transduction cascade). Distinguish different types of signaling molecules. Explain the role of signaling molecules in converting extracellular signals into cellular responses (in signal transduction). Distinguish different classes of receptors (nuclear, cytosolic and plasma membrane) and the type of signaling molecules they bind and respond to. Explain the role of intracellular transducers in relaying biosignals. Understand the concept of signal amplification and its role in the transmission of intracellular signals. Explain how nuclear and cytosolic receptors produce a cellular response. Give examples of pharmacotherapies that target nuclear and cytosolic receptors. Explain how ion channel receptors (ligand-gated ion channel receptors) work.  The Cellular Internet Cell-to-cell communication is essential for multicellular organisms. A signal transduction pathway is a series of steps by which a signal on a cell’s surface is converted into a specific cellular response. Signal transduction pathways convert signals on a cell’s surface into cellular responses. A mammalian cell depends on multiple extracellular signals Basic Elements of a Signal Transduction Pathway Signaling molecules (ligands) bring the signal from the extracellular environment Receptors – receive specific signals by binding signaling molecules Transducers - transmit the message from the receptor to the effector target Intracellular effectors Cellular response – change in cell metabolism or function.  Local and Long-Distance Signaling Cells in a multicellular organisms communicate by chemical messengers. Animal and plant cells have cell junctions that directly connect the cytoplasm of adjacent cells. In local signaling, animal cells may communicate by direct contact. In many other cases, animal cells communicate using local regulators, messenger molecules that travel only short distances. In long-distance signaling, mammalian cells use chemicals called hormones.  Modes of cell-cell signaling Direct: cell-cell or cell-matrix Indirect: secreted molecules. Types of Chemical Signaling Images are created by Heather Ng-Cornish  Local signaling Long-distance signaling Target cell Electrical signal Endocrine cell Blood along nerve cell vessel triggers release of neurotransmitter Neurotransmitter Secreting Secretory diffuses across cell vesicle synapse Hormone travels in bloodstream to target cells Local regulator diffuses through Target cell Target extracellular fluid is stimulated cell Paracrine signaling Synaptic signaling Hormonal signaling A.Endocrine signaling. The signaling molecules are hormones secreted by endocrine cells and carried through the circulation system to act on target cells at distant body sites. B.Paracrine signaling. The signaling molecules released by one cell act on neighboring target cells. C.Autocrine signaling. Cells respond to signaling molecules that they themselves produce (response of the immune system to foreign antigens, and cancer cells).  Stages of Cell Signaling Earl W. Sutherland discovered how the hormone epinephrine acts on cells. (Nobel Prize for Physiology and medicine 1971). Sutherland suggested that cells receiving signals went through three processes: Reception Transduction Response A signal is sent which is received by the cell. This signal is interpreted by that cell that receives it. Finally, the cell responds to the signal.  Three Steps in Cell Signaling Target organ specificity is the result of specific receptor molecules for the hormone, either on the plasma membrane surface, nucleus or in some cases in the cytoplasm, of cells in the target organ. 1) Reception 2) Transduction 3) Response Reception: A signal molecule binds to a receptor protein, causing it to change shape. The binding between a signal molecule (ligand) and receptor is highly specific. A conformational change in a receptor is often the initial transduction of the signal. Most signal receptors are plasma membrane proteins.  Transduction: Cascades of molecular interactions relay signals from receptors to target molecules in the cell Transduction usually involves multiple steps Fine-Tuning of Multistep pathways can amplify a the Response signal: A few molecules can produce 1- Amplifying the signal (and thus the response) a large cellular response 2- Specificity of the response 3- Overall efficiency of Multistep pathways provide more response, enhanced by scaffolding proteins opportunities for coordination and regulation  Response: This is the end of the line for a signal brought to the target cell by a signal molecule and goes into action as the cellular response. INTEGRATE: Multiple receptors FEEDBACK: As the signal moves DISTRIBUTE: At some point, it’s feed into a single through the cascade of responses, probable that an enzyme will be “downstream” response. In later events can influence earlier activated whose function is to some cases, both signals will components of the pathway activate many different proteins. be required for the response to (feedback loop). Feedback loops The proteins that are being acted occur, while in other cases, can be either positive (i.e., later upon will have a wide variety of either signal can cause the events help further activate earlier functions (e.g., transcription response in question, steps) or negative (i.e., later events factors). This is how the signal is regardless of whether the stop earlier events from continuing). distributed so that cellular function other is present. changes.  Termination : Stopping or slowing down the signal Inactivation mechanisms are an essential aspect of cell signaling. When signal molecules leave the receptor or it is degraded, the receptor reverts to its inactive state. Generic Signaling Cascade Reception and Relay Transduce and Amplify Integrate Feedback Distribute Response Deactivation Reception: A signal molecule binds to a receptor protein, causing it to change shape.  Receptors: Most water-soluble signal molecules bind to specific sites on receptor proteins in the plasma membrane The main types of membrane (cell surface) receptors: vG-protein-coupled receptors vReceptor tyrosine kinases vIon channel receptors Found in the cytosol or nucleus of target cells Small or hydrophobic chemical messengers can readily cross the membrane and activate receptors vExamples of hydrophobic messengers are the steroid and thyroid hormones of animals. Activated hormone-receptor complexes can act as a transcription factor, turning on specific genes  RECEPTOR TYROSINE KINASES ION CHANNEL RECEPTORS G-PROTEIN COUPLED RECEPTORS  Testosterone signaling Hormone EXTRACELLULAR Conformational changes in (testosterone) FLUID The steroid hormone testosterone IGF1 Receptor for its activation passes through the plasma membrane. Plasma membrane Testosterone binds Receptor to a receptor protein protein in the cytoplasm, Hormone- activating it. receptor complex The hormone- receptor complex enters the nucleus and binds to specific genes. DNA mRNA The bound protein stimulates the transcription of the gene into mRNA. NUCLEUS New protein The mRNA is translated into a https://doi.org/10.7554/eLife.04909 CYTOPLASM specific protein. Transduction: Cascades of molecular interactions relay signals from receptors to target molecules in the cell Signal Amplification Enzyme cascades amplify the cell’s response At each step, the number of activated products is much greater than in the preceding step  Signal The Specificity of Cell Signaling molecule Receptor Relay Different kinds of cells have different molecules collections of proteins Response 1 Response 2 Response 3 These differences in proteins give Cell A. Pathway leads Cell B. Pathway branches, each kind of cell specificity in to a single response leading to two responses detecting and responding to signals The response of a cell to a signal depends on the cell’s particular collection of proteins Activation or inhibition Pathway branching and “cross-talk” further help the cell coordinate Response 4 Response 5 incoming signals Cell C. Cross-talk occurs Cell D. Different receptor between two pathways leads to a different response  Protein Phosphorylation and Dephosphorylation Signal molecule In many pathways, the signal is Receptor Activated relay transmitted by a cascade of protein molecule phosphorylation. Inactive protein kinase Active 1 protein Phosphatase enzymes remove the kinase 1 Ph Inactive os phosphates. protein kinase ATP ADP ph Active P or 2 protein yla PP kinase tio Pi This phosphorylation (kinases) and n 2 ca Inactive sc protein kinase ATP ADP ad Active P dephosphorylation (phosphatases) e 3 protein PP kinase Pi system acts as a molecular switch, Inactive ATP 3 protein ADP P turning activities on and off. PP Active protein Cellular response Pi  Small Molecules and Ions as Second Messengers Second messengers are small, nonprotein, water-soluble molecules or ions. The extracellular signal molecule that binds to the membrane is a pathway’s “first messenger” Second messengers can readily spread throughout cells by diffusion Second messengers participate in pathways initiated by G-protein-coupled receptors and receptor tyrosine kinases  First messenger Cyclic AMP (signal molecule such as epinephrine) Adenylyl G protein cyclase Cyclic AMP (cAMP) is one of the most widely used second G-protein-linked GTP receptor messengers ATP Adenylyl cyclase, an enzyme in cAMP Second messenger the plasma membrane, converts ATP to cAMP in Protein kinase A response to an extracellular signal Cellular responses  EXTRACELLULAR FLUID Plasma Calcium ions membrane Ca2+ pump ATP Mitochondrion Calcium ions (Ca2+) act as a second messenger in many pathways Nucleus Calcium is an important second CYTOSOL messenger because cells can regulate its concentration Ca2+ pump A signal relayed by a signal Endoplasmic reticulum (ER) transduction pathway may trigger ATP Ca2+ pump an increase in calcium in the cytosol Key High [Ca2+] Low [Ca2+]  EXTRACELLULAR Signal molecule Inositol FLUID (first messenger) Triphosphate (IP3) G protein DAG GTP G-protein-linked PIP2 Pathways leading to the receptor Phospholipase C IP3 (second release of calcium messenger) involve inositol IP3-gated calcium channel triphosphate (IP3) and diacylglycerol (DAG) as Various Cellular Endoplasmic Ca2+ proteins responses second messengers reticulum (ER) activated Ca2+ (second CYTOSOL messenger)  Many other signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus The final activated molecule may function as a transcription factor  ION CHANNEL RECEPTORS Often involved in rapid synaptic (paracrine) signaling. Ligand binding activates receptor almost instantaneously. Effects include change in the plasma membrane potential due to ion influx (Ca2+, Na+ influx - membrane depolarization , Cl- influx – membrane hyperpolarization) and generation of electrical impulse. Ligand Examples: acetylcholine, neurotransmitters, GABA, glutamate, inositol triphosphate (IP3), physical stimuli  G PROTEIN-COUPLED RECEPTORS (Serpentine or 7 Trans-Membrane Receptors) 1. When the receptor binds to its ligand, it undergoes a conformation change and binds to the α/β/γ complex (attached to the GPCR on the cytosolic face of the plasma membrane.) This causes the GDP to be released from the α-subunit and get replaced by GTP. 2. The binding of the GTP molecule causes a conformation change in the G-protein complex, and the β/γ subunits are released from Gα. 3. The separated G-protein subunits (Gα and Gβγ) are now considered active and activate their appropriate downstream targets.  G PROTEIN-COUPLED RECEPTORS (Activation) Inactive receptor does 1. NOT have a binding site for trimeric G protein. 2. 1. Active receptor binds to Gα subunit. 3. 2. This binding induces GDP to be replaced by GTP and activates trimeric G protein. Gα dissociates from Gβγ, thus active G protein dissociates from the receptor. 3. Active Gα subunit binds to and activates an effector protein (enzyme or ion channel)  ON – OFF switching of trimeric G protein As the G-proteins are activated and move away from the receptor, the lipid tails keep them associated with the interior surface of the plasma membrane. They will be able to diffuse laterally through the fluid membrane until they meet proteins that they can bind to and activate. Eventually the G-proteins will deactivate themselves. The Gα subunit (GTPase) will slowly hydrolyze the GTP so that it is converted back into GDP. This inactivates the Gα subunit, causing all three G-proteins to return to their inactive state. Because the activation of the G-proteins is dependent on the presence of GTP in the Gα subunit, this provides a built-in system for shutting down the response. The GTP is short lived, thus the response lasts only as long as the GTP does. However, there may be many cycles of activation and deactivation of G-proteins during a single response.  G PROTEIN-COUPLED RECEPTORS (Activating the effector) The activated trimeric G protein activates an enzyme or channel transforming the extracellular signal into the intracellular response.  Enzyme-Coupled Receptors RECEPTOR RECEPTOR TYROSINE PROTEIN KINASES PHOSPHATASES RECEPTOR SER/THR KINASES With intrinsic phosphorylation capacity, meaning that they can act on themselves or their dimerized neighbor, which is activated upon ligand binding. No intrinsic enzymatic activity but will be closely associated with a kinase that is activated upon ligand binding.  RECEPTOR TYROSINE KINASES Example of a receptor tyrosine kinases activation: The ligand is a dimer and binds to the receptor on the extracellular side, causing two monomeric receptors to dimerize, cross phosphorylate, and activate. Phosphorylation creates docking sites, allowing for recruitment of other enzymes and downstream signaling targets.  RECEPTOR TYROSINE KINASES In the absence of the ligand, the receptor exists as a monomer, which is inactive form of the receptor. When the ligand (e.g.,hormone, growth factor) binds to the extracellular domain, two monomers form a dimer. Dimerization of the receptor is followed by cross-phosphorylation of both cytosolic domains and activation of the receptor. Some other receptors exist as dimers even in the absence of the ligand. The receptor is always present as a dimer. Binding of the ligand results in cross-phosphorylation of the cytosolic domains of the receptor and receptor activation.  RECEPTOR TYROSINE KINASES RTK cascade can be activated Ras-dependent pathway Ras-independent pathway (no second messengers) (second messengers) Proliferation Metabolism Differentiation Transport Functions Cell Survival Proliferation Stress Responses Differentiation Cell Survival  GPCR signal attenuation RTK signal attenuation Ligand dissociation from the receptor. Receptor phosphorylation, 𝜷-arrestin binding and Ligand-sequestration and binding inhibition. internalization (endocytosis). Dephosphorylation via protein tyrosine phosphatases (PTP). GTP hydrolysis by G𝜶 subunit. Inhibition of RTK autophosphorylation. Hydrolysis of the phosphodiester bond in cAMP. Inhibitory proteins that counteract downstream signaling. IP3 dephosphorylation to inositol. Ligand-induced receptor ubiquitination and degradation. Figure by Fernanda Ledda Cell Signaling in Physiology and Medicine  Biosignaling in Human Disease and Drug Development  Second Messengers are molecules that relay signals from receptors to target molecules. Low amount in resting state. Cyclic nucleotides: Short lived molecules. cAMP Their elevated concentration leads to cGMP rapid alteration in the activity of one or Membrane lipid derivatives more cellular enzymes. PIP2 Their synthesis and degradation are PIP3 regulated. IP3/DAG Removal or degradation of second messenger terminate the cellular Calcium response. NO /CO/H2S  Cyclic Nucleotides as second messengers cAMP cGMP Ligands: Hormones, ACH,… Ligands: NO, ANP (natriuretic Primary Effector: Adenylyl Cyclase peptide)… cAMP activates Protein Kinase A Primary Effector: Guanylyl Cyclase (PKA) which then phosphorylates, Many cells contain a cGMP- usually the OH group of Ser or Thr stimulated protein kinases that of the target proteins in cytosol and contains both catalytic and nucleus. regulatory subunits. Some of the effects of cGMP are mediated through Protein Kinase G (PKG). cGMP serves as the second messenger for nitric oxide.  Nitric Oxide PI derived second (NO ) messengers NO is a free radical gas. Phosphatidylinositol (PI) is a It can diffuse across membranes negatively charged phospholipid. and alter intracellular target Inositol can be phosphorylated to enzymes. form PIP, PIP2, PIP3 as second It is unstable so its effect are local. messengers. When acetylcholine is released from PIP2 can be hydrolyzed by phospho the terminus of nerve cells in the lipase (PLC) to yield another set of blood vessel walls, the endothelial second messengers: cells are stimulated to produce NO. Diacylglycerol (DAG) NO then increase the synthesis of Activates PKC cGMP, activate PKG for platelet Inositol -1,4,5-triphosphate (IP3) inhibition, vasodilation, etc. Opens Ca2+ channels (endothelium-derived relaxing factor – EDFR)  Activation of Protein Kinase G via cGMP Protein Kinase G  Protein Kinase G Promotes the opening of K+ CONTRACTION channels and inhibits Ca2+ channel in the plasma membrane, leading to cell hyperpolarization. Indirectly stimulates Ca2+-ATPase that pumps Ca2+ from the cytosol into the endoplasmic reticulum, thus lowering calcium levels in the cytosol and increasing calcium levels in ER. Cytosolic Ca2+ RELAXATION Blocks activity of phospholipase levels are down C and generation of inositol triphosphate reducing the release of calcium ions stored in the endoplasmic reticulum.  Less cGMP Less Relaxation 5’ GMP cGMP-dependent phosphodiesterase-5 VASODILATION 5’ GMP cGMP-dependent phosphodiesterase-5 Activation of Protein Kinase A via cAMP  Activation of Protein Kinase C via Ca2+ and DAG 1. Receptor (GPCR or RTK) activation following ligand binding leads to phospholipase C (PLC) activation. 2. Active PLC cleaves phosphatidylinositol 4,5- bisphosphate (PIP2) into IP3 and diacylglycerol (DAG). 3. IP3then stimulates calcium release from the endoplasmic reticulum. 4. Calcium allows PKC to bind with DAG. 5. PKC is now activated. Lithium targets second messenger systems that modulate neurotransmission. Lithium affects the adenyl cyclase and PI pathways, as well as PKC, and may serve to decrease excessive excitatory neurotransmission from the dopamine receptor (G-protein coupled receptor). Lithium can also inhibit MARCKS, GSK3, two other protein phosphatases, which are excessively activated under chronic stress conditions, such as mania.  Insulin Signaling IRS pathway Activation of kinases dependent upon the heterodimeric (p85/p110) PI3K, such as Akt, also known as protein kinase B (PKB); Akt modulates enzyme activities that, besides affecting NO generation and apoptosis, control glucose, lipid, and protein metabolism. RAS independent metabolic arm MAPK signaling cascade PTEN Grb2/Sos pathway leads to activation (phosphatidylinositol-3,4,5- of Ras signaling, affecting cell triphosphate 3-phosphatase) proliferation and apoptosis. In view of their mitogenic nature, these can be characterized as “growth signal” RAS dependent effects. mitogenic arm McLoughlin et al., 2018 Drugs Targeting G-Protein Coupled Receptors (GPCRs) GPCRs are the largest family of membrane receptors. Involved in signal transduction by interacting with G-proteins. Regulate key physiological functions (e.g., heart rate, neurotransmission, immune response). 1. Drug Classes: 2. Beta-blockers (GPCR: Adrenergic receptors) Examples: Metoprolol, Propranolol (Hypertension, arrhythmias, heart failure 3. Opioid Analgesics (GPCR: Opioid receptors) Examples: Morphine, Fentanyl (Pain management) 4. Antihistamines (GPCR: Histamine H1 receptors) Examples: Loratadine, Cetirizine (Allergies) 5. Angiotensin II Receptor Blockers (GPCR: AT1 receptor) Examples: Losartan, Valsartan (Hypertension, heart failure) 6. Serotonin Agonists/Antagonists (GPCR: Serotonin receptors) Examples: Sumatriptan (agonist-migrane), Ondansetron (antagonist – N/V) Mechanism of Action: Drugs either activate (agonists) or inhibit (antagonists) GPCRs. Modulate intracellular signaling cascades through second messengers (e.g., cAMP, Ca²⁺). Drugs Targeting Receptor Tyrosine Kinases RTKs are transmembrane receptors with intrinsic kinase activity. Play a critical role in cell proliferation, survival, and differentiation. Commonly dysregulated in cancer. Drug Classes & Examples: 1.Monoclonal Antibodies Bind extracellular domain of RTKs 2.Tyrosine Kinase Inhibitors (TKIs) Inhibit kinase activity 3.Multi-target TKIs Inhibit multiple RTKs Mechanism of Action: Monoclonal antibodies block ligand binding, preventing receptor activation. TKIs inhibit autophosphorylation of RTKs, blocking downstream signaling pathways essential for cancer cell survival and proliferation.