CELL SIGNALING AND MOTILITY BIOL 3373 Lecture 3

CELL SIGNALING AND MOTILITY (BIOL 3373) Lecture 3 1

WHAT IS THE CELL SIGNALING? How cells receive and respond to signals from their surroundings ü On one hand, cell signaling regulates gene expression and controls the cell fate (proliferation, motility, differentiation and programmed cell death, or apoptosis). ü On the other hand, cell signaling allows for the organization of cells into tissues, which, in turn, generate organs. In addition, cell signaling is essential for the maintenance of cells, tissues and organs. 2

CELL SIGNALING q Communication among cells is referred as intercellular signaling. q Cells communicate with each other through signaling molecules. q Signaling molecules could be: ü proteins, small peptides, amino acids, ü nucleotides, ü steroids, retinoids, fatty acid derivatives, ü nitric oxide, carbon monoxide 3

Forms of signaling molecules : Steroid hormones are a group of hormones that belong to the class of chemical compounds known as steroids. All steroid hormones are derived from cholesterol. They are transported through the bloodstream to the cells of various target organs where they carry out the regulation of a wide range of physiological functions. These hormones often are classified according to the organs that synthesize them. The adrenal cortex produces the adrenocortical hormones, which consist of the glucocorticoids and the mineralocorticoids. Glucocorticoids such as cortisol control many metabolic processes, including the formation of glucose and the deposition of glycogen in the liver. Mineralocorticoids such as aldosterone help maintain the balance between water and salts in the body, predominantly exerting their effects within the kidney. The androgens are the male sex hormones, responsible for the development and maintenance of reproductive function in the male. The principal androgen, testosterone, is produced by the testes. Estrogens are female sex hormones. They are secreted mainly by the ovaries. Estradiol is the most potent of the estrogens promote the development of the primary and secondary female sex characteristics. Progestins, the most important of which is progesterone, are the other type of female sex hormone and are named for their role in maintaining pregnancy (pro-gestation). 4

Forms of signaling molecules- Gas - NO Nitric oxide (NO) is a gas molecule produced and released by endothelial cells (signaling cells) and rapidly diffuses across the membranes. NO binds to an enzyme inside the smooth muscle cells (target cell), inducing cell relaxation 5

Forms of signaling molecules Neurotransmitters are chemicals produced and released by neurons. Neurotransmitters travel across the synapse and allow communication between neurons. 6

Forms of signaling molecules Neurotransmitters 7

Forms of signaling molecules Peptide Hormones and Growth Factors 8

CELL SIGNALING q Cells that produce and release the signaling molecules are signaling cells. q Cells that receive the signal are target cells q Targets cells posses specific receptors that recognize signaling molecules. receptor signaling cell signaling molecules target cell 9

CELL SIGNALING Depending on the distance that the signaling molecule has to travel, we can talk about four types of signaling: Contact- dependent signaling requires cells to be in direct membrane contact. The signaling molecule remains bound to the surface of the signaling cells. It influences only cells (target cells) contact it via a specific protein receptor. Contact-dependent signaling is especially important during development and immune response from Alberts et al. , Molecular Biology of the Cell. 6 th edition. 10

CELL SIGNALING In paracrine signaling the signaling molecules are local mediators and affects only target cells in the proximity of the signaling cell. An example is the conduction of an electric signal from one nerve cell to another or to a muscle cell. In this case the signaling molecule is a neurotransmitter. from Alberts et al. , Molecular Biology of the Cell. 6 th edition. 11

CELL SIGNALING In autocrine signaling cells respond to molecules they produce themselves. So signaling cells are also target cells. Examples include cancer cells as they can produce signals that stimulate their own survival and proliferation. autocrine signaling cell = target cell from Alberts et al. , Molecular Biology of the Cell. 6 th edition. 12

CELL SIGNALING Large multicellular organisms use long- range signaling that coordinate the behavior of the cells in remote part of the body. Synaptic signaling is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which can be located far away from the neuron cell body. from Alberts et al. , Molecular Biology of the Cell. 6 th edition. 13

CELL SIGNALING In endocrine signaling molecules (hormones) are produce by endocrine cells and sent through the blood stream to distant cells 14

The Three Stages of Cell Signaling • Earl W. Sutherland discovered how the hormone epinephrine acts on cells • Sutherland suggested that cells receiving signals went through three processes: – Reception – Transduction – Response 15

Overview of cell signaling Cell signaling consists of 3 stages EXTRACELLULAR FLUID 1 Reception CYTOPLASM Plasma membrane 2 Transduction 3 Response Receptor Activation of cellular response Relay molecules in a signal transduction pathway Signal molecule Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Three Stages of Cell Signaling: 1 Reception EXTRACELLULAR FLUID CYTOPLASM Plasma membrane 1 Reception Receptor The receptor and signaling molecules fit together (lock and key model) Signaling molecule binds to the receptor protein at the cell surface 17

1 - 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 18

RECEPTORS IN THE PLASMA MEMBRANE 19

Intracellular Receptors • Some receptor proteins are intracellular, found in the cytosol or nucleus of target cells • Small or hydrophobic chemical messengers can readily cross the membrane and activate receptors • Examples of hydrophobic messengers are the steroid and thyroid hormones of animals • An activated hormone-receptor complex can act as a transcription factor, turning on specific genes 20

Hydrophobic signaling molecules can cross the plasma membrane and bind to nuclear receptors 21

Hormone (testosterone) EXTRACELLULAR FLUID Plasma membrane Receptor protein Hormonereceptor complex The steroid hormone testosterone passes through the plasma membrane. Testosterone binds to a receptor protein in the cytoplasm, activating it. The hormonereceptor complex enters the nucleus and binds to specific genes. DNA The bound protein stimulates the transcription of the gene into m. RNA NUCLEUS New protein The m. RNA is translated into a specific protein. CYTOPLASM

The nuclear receptor superfamily Nuclear receptors are inactive without signaling molecules (ligands) When the nuclear receptor interacts with the specific ligand, it becomes activate and induces the transcription of target genes 23

Three Stages of Cell Signaling: 2 Transduction CYTOPLASM EXTRACELLULAR FLUID Plasma membrane 1 Reception 2 Transduction Receptor 2 nd Messenger! Relay molecules in a signal transduction pathway Signaling molecule The signal from the receptor is converted into a intracellular message that produces a cellular response. 24

SIGNAL TRANSDUCTION: The study of the molecular circuits responsible for the generation of a cellular response after the delivery of a signal. Includes a NETWORK of molecular and cellular events conveying a SIGNAL from the outside to the inside of the cell. 25

SIGNAL TRANSDUCTION Signaling molecules (ligands) bind to receptors on target cells. After the binding with the ligand, the receptor activates one or more intracellular signaling within the target cells, that involves several proteins ( transducer Protein). The intracellular signaling modulates the activity of target proteins (also known as effector proteins) thereby the behavior of the cells. from Alberts et al. , Molecular Biology of the Cell. 6 th edition. 26

Three Stages of Cell Signaling: 3 Response CYTOPLASM EXTRACELLULAR FLUID Plasma membrane 1 Reception 2 Transduction 3 Response Receptor Activation of cellular response Relay molecules in a signal transduction pathway Signaling molecule Cellular responses can be different and complex (i. e. change in gene expression, cell motility, cell growth, cell differentiation, cell death), depending on cell types. 27

Cytoplasmic and Nuclear Responses • The response may occur in the cytoplasm or in the nucleus • Many signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus • The final activated molecule in the signaling pathway may function as a transcription factor 28

Cytoplasmic and Nuclear Responses • 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 29

Fine-Tuning of the Response • There are four aspects of fine-tuning to consider – signal Amplification (and thus the response) – Specificity of the response, – Efficiency of response, – Initiation of the signal, – Termination of the signal, 30

Signal Amplification ü At each step of many signal transduction pathways, the number of activated participants in the pathway increases. This is referred as signal amplification, figure 1 ü For example, one epinephrineactivated GPCR activates 100 s of Gas-GTP complexes, which in turn activate 100 s of adenylyl cyclase molecules, that each produce hundreds of c. AMP molecules, and so on. The overall amplification associated with epinephrine signaling is estimated to be ~108 -fold (figure 1) 31

Specificity of the response • Different kinds of cells have different collections of proteins • These differences in proteins give each kind of cell specificity in detecting and responding to signals • The response of a cell to a signal depends on the cell’s particular collection of proteins • Pathway branching and “cross-talk” further help the cell coordinate incoming signals 32

Specificity of the response Signal molecule Receptor Relay molecules Response 1 Cell A. Pathway leads to a single response Response 2 Response 3 Cell B. Pathway branches, leading to two responses Activation or inhibition Response 4 Cell C. Cross-talk occurs between two pathways Response 5 Cell D. Different receptor leads to a different response 33

THE SAME SIGNAL CAN INDUCE DIFFERENT RESPONSES IN DISTINCT CELL TYPES 34

Efficiency of response depends on: The expression of specific receptors; The bioavailability of transducer molecules: Expression levels/half-life Localization within the cell Modality of activation/inactivation The bioavailability of effector molecules: cytoskeletal elements (morphological changes) transcription factors (changes in gene expression) proteolytic enzymes (cell death) 35

INITIATION OF SIGNAL: RECEPTOR ACTIVATION/ INACTIVATION For EACH signaling pathway there is a common feature that defines a sequence of events: üThe receptor is in an INACTIVE STATE in the absence if the signal üWhen the signal arrives, it BINDS to the receptor, which MODIFIES its conformation üThis change in the receptor ACTIVATES other molecules (i. e. a downstream signaling cascade) 36

Termination of the Signal: • Inactivation mechanisms are an essential aspect of cell signaling • When signal molecules leave the receptor, the receptor reverts to its inactive state. 37

THE SIGNALING PATHWAY VIA G-PROTEIN 38

G-protein coupled receptors G- protein coupled receptors consists of 3 components: 1. trans-membrane receptors (GPCRs) known also as serpentine receptors. 2. G proteins: Guanine nucleotide-binding proteins, trimeric G protein. 3. Effectors: Effectors can be different : adenylyl cyclase or Phosphodiesterase (PDE) or phospholipase C-β (PLC- β) or ion Channel. Receptor adenylyl cyclase PDE PLC- β ion Channel G protein Effector 39

These effectors in turn regulate the intracellular concentrations of secondary messengers, such as c. AMP, c. GMP, diacylglycerol (DG), IP 3, sodium (Na+), potassium (K+) or calcium cations (Ca 2+), which ultimately lead to a physiological response, usually via the downstream regulation of gene transcription. adenylyl cyclase PDE c. AMP PLC- β c. GMP ion Channel Na+, DG K+ Ca 2+ IP 3 Second messengers 40

G-protein coupled receptors Several types of receptors are associated with G proteins, such as hormones, neurotransmitters, growth factors, glycoproteins, cytokines, odorants and photons 41

G-protein-coupled receptors Extracellular loops NH 2 N -Terminal chain Membrane VII VI V IV III II I Transmembrane helix G-Protein binding region HO 2 C C -Terminal chain Variable intracellular loop Intracellular loops Single protein composed of 7 transmembrane domains with an extracellular amino terminus and an intracellular carboxyl terminus. Intracellular C terminus changes conformation in response to a stimulus and this results in changes in the ability to recruit G proteins. 42

G-protein G proteins are composed by 3 subunits, α, β and γ. The α subunit contains the GDP binding site as well as the GTP hydrolysis domain. 43

ADENYLYL CYCLASE : EFFECTOR OF GPCR First messenger (signal molecule such as epinephrine) Adenylyl cyclase G protein G-protein-linked receptor GTP ATP c. AMP Second messenger Adenylyl cyclase is a multipass transmembrane protein that converts ATP to form cyclic AMP (c. AMP). Protein kinase A Cellular responses 44

Cyclic AMP (c. AMP) Phosphodiesterase Adenylyl cyclase ATP Pyrophosphate P Pi Cyclic AMP H 2 O AMP ü The Adenylyl Cyclase catalyzes the reaction that leads to the production of c. AMP. ü c. AMP is synthesized from ATP through a cyclization reaction that removes two phosphate group as pyrophosphate. ü c. AMP is an unstable molecule because it is soon hydrolyzed to AMP by specific Phosphodiesterases. 45

Activation of G-protein 1. The binding of the ligand to the receptor changes the receptor conformation. 2. The new receptor conformation allows the binding of the G protein. 3. When the G protein assembles to the receptor it alters its own conformation, therefore the GDP binding site within the α subunit is distorted and GDP is released. Ligand 1 2 3 Cell membrane Receptor ß g a G Protein ß g a GDP Binding site for G-protein opens 46 GTP

Activation of G-protein 4. The binding of GTP to the α subunit promotes the closure of the nucleotide binding site within the α subunit. 5. Therefore the α subunit changes its conformation and 6. separates from both the β-γ dimer and the receptor. ü This process is active while the ligand is bound to the receptor ü One ligand can activate several G protein: Signal amplification 4 ß g a GTP binds 6 5 g ß g a Fragmentation and release ß a GTP α subunit changes conformation 47

Activation of G-protein 7. The α-GTP subunit interacts with the Adenylyl cyclase 8. Adenylyl cyclase becomes active and converts ATP in c. AMP. 9. The cycle is completed when GTP is hydrolyzed to GDP within the α subunits. This causes the re-association of α subunit with β-γ dimer and the binding of G-protein to the receptor, which terminates the cycle. Binding site for a subunit 7 8 9 GTP hydrolysed to GDP catalysed by a subunit Binding P ATP cyclic AMP Active site (closed) Active site (open) Signal transduction (con) a Subunit recombines with b-g dimer to reform inactive G protein. GTP GDP as-subunit Adenylyl cyclase 48

Activation of protein Kinase A (PKA) o. The target proteins of c. AMP vary Adenylate cyclase depending on cell types, however the best characterized c. AMP target is ATP cyclic AMP Activation the PROTEIN KINASE A (PKA). o PKA phosphorylates serine or PKA threonine residues on target proteins P Enzyme (inactive) thereby regulating their activity. Enzyme (active) P Target Protein o Among the target proteins of PKA the phosphodiesterases are responsible to lower c. AMP concentration thus keeping PKA activation short and localized. 49

Target Proteins of protein Kinase A (PKA) Examples of a signaling pathway mediated by c. AMPPKA include the activation of transcription regulator, the CRE binding protein (CREB). PKA phosphorylates CREB on a single serine. Active phosphorylated CREB (p-CREB) recruits the coactivator CREB-binding protein (CBP) (not known) to the regulatory sequence (CRE) present in many genes (i. e. somatostatin gene) that are activated by c. AMP. CREB signaling plays an important role in the learning and memory process in the brain. 50

Activation of protein Kinase A (PKA) PKA is a complex protein consisting of 2 regulatory subunits and two catalytic domains. The regulatory domains bind to the cytoskeleton or membrane organelle, thereby tethering PKA in specific subcellular compartment. Four c. AMP molecules bind to the PKA regulatory domains, causing their dissociation from the protein complex and the consequent activation of the catalytic subunits. 51

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Smell depends on GPCRs that regulate cyclic-nucleotide-gated ion channels Olfactory receptor neurons line on the nose. These cells use GPCRs called olfactory receptors to recognize odors; the receptors are displayed on the modified cilia that extend from each cell. Olfactory receptor neurons 53

Sensory transduction in olfaction Odorant molecules bind to olfactory receptors activating a G protein, which in turn activates adenylyl cyclase. Second-messenger systems are activated, Na+ (sodium) and Ca 2+ (calcium) or Ca 2+ channels are opened, and the cilia are depolarized. This depolarization iniziates a nerve implulse thart travels alomg its axon to the brain 54

Vision depends on GPCRs that regulate cyclic-nucleotide-gated ion channels 55

Photo- transduction in photoreceptor cells In the retina the rod cells contains a pigment protein, rhodopsin, which is a GPCRs associated with the G protein, Transducin. t gh i L Na+ Rod cells Signal = light GMP c. GMP gated ion channel G-protein coupled receptor= Rhodopsin (Rods) G protein = transducin enzymatic activity = phosphodiesterase (PDE) second messenger = DECREASE in GMP Na+ channel closure 56

Photo- transduction in photoreceptor cells In dark condition the rhodopsin are inactive c. GMP-gated ion channels are opened the rod cell are depolarized high release of neurotransmitters In light condition rhodopsin absorbs light and activates transducing c. GMP decrease c. GMPgated ion channels become closed reduced the release of neurotransmitters. 57

Amplification of Photo- transduction one rhodopsin activates 500 s of G -GTP complexes, which in turn activate 500 s of GMP phosphodiesterase , that each produce 105 of c. AMP molecules, and so on. The overall amplification associated with rhodopsin signaling result in the alteration of membrane potential of 1 m. V 58

Stimulatory G-protein and Inhibitory G protein Stimulatory G proteins (Gs) increase the c. AMP concentration and activate PKA. However several extracellular signals activate a different class of G proteins known as Inhibitory G proteins (Gi). Inhibitors G protein blocks the Adenylyl Cyclase ( AC) activity thereby reducing c. AMP levels. 59

Epinephrine-induced breakdown of glycogen. Reception Binding of epinephrine to G-protein-linked receptor (1 molecule) Transduction Inactive G protein ü Best characterized example of Gs protein coupled receptor is the epinephrine receptor. Active G protein (102 molecules) Inactive adenylyl cyclase Active adenylyl cyclase (102) ü The binding of epinephrine to Gprotein-linked receptor triggers a signaling pathway leading to the release of glucose from glycogen, which takes places in hepatocytes (liver cells). ATP Cyclic AMP (104) Inactive protein kinase A Active protein kinase A (104) Inactive phosphorylase kinase Active phosphorylase kinase (105) Inactive glycogen phosphorylase Active glycogen phosphorylase (106) Response Breakdown of glycogen to glucose monomers by phosphorolysis in liver and muscle cells Glycogen Glucose-1 -phosphate (108 molecules) 60

Stimulatory G-protein and Inhibitory G protein Both Stimulatory G proteins (Gs) and Inhibitory G proteins (Gi) are targets for bacterial toxins: Cholera toxin stimulates Gs: ü catalyzes ADP ribosylation of the α subunit of Gs and blocks its GTPase activity thereby compromising the GTP hydrolysis. As a result, α subunit of Gs becomes permanently “active” causing a persistent stimulation of adenylate cyclase and elevated c. AMP large efflux of water and Cl- in the gut that causes diarrhea. 61

Stimulatory G-protein and Inhibitory G protein Pertussin toxin stimulates Gi: ü catalyzes ADP ribosylation of the α subunit of Gi keeping it in the inactive GDP bound state. 62

IN CLASS PRESENTATION ON FEBRUARY 11, 2019 Establish 4 team groups (12 max people per group ): Each student should join a group of choice. (Each of you is free to join a group of interest but, if you prefer, I can make up the group) Each team needs to: 1. Choose 1 out 4 scientific articles I have sent for today class; 2. Read the chosen article and make a 20 min presentation to be delivered for in class presentation on February 11; 3. The presentation should be shared among team members; 4. Be prepared to answer in class questions from the professor and your peers. Please send to me (antonio. giordano@ temple. edu) the following: § Name of the students for each group and the paper chosen by February 7 §PPT presentation along with information for each group by 12 AM on February