MB207 Molecular Cell Biology Cell Communication

2 General principles of cell communication All cells have some ability to sense and respond to specific chemical signals. → Intercellular communication. Signal is transmitted by regulatory chemical messengers and receptors that are located on the receptors located on the surfaces of the cells that may be quite distant from the secreting cells. Three general categories of chemical signaling: i) Cytoplasmic connections between cells ii) Cell-to-cell contact-mediated signaling iii) Free diffusion between cells - Distants (hormones) - Adjacent cells (within interstitial space)

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4 Autocrine signals → Signals that act on the same cell that produces them. Paracrine signals → Signals that are released locally, where they diffuse to act at short range on nearby tissues. Endocrine signals → Signals that are produced at great distances from their target tissues and are carried by the circulatory system to various sites in the body.

5 Local diffusion

6 Long-distance diffusion

7 1) Reception of extracelluar signal by cell. 2) Transduction of signal from outside of cell to inside of cell. → Often includes multiple steps. 3) Celluar response. → Response is initiated and/or occurs entirely within receiving cell. Three stages of signal transduction

8 2a. Transduction 2b. Transduction 2c. Transduction 2d. Transduction 1. Reception 3. Response

9 2a. Transduction 2b. Transduction 1. Reception 3. Response

10 More than one response can result form the reception of a single ligand

11 Cell Communication - reception Communication between cells requires: i) ligand: the signaling molecule ii) receptor protein: the molecule to which the receptor binds -may be on the plasma membrane or within the cell Receptors are defined by location: i) Cell surface receptors → located on the plasma membrane. ii) Intracellular receptors → located within the cell.

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13 Intracellular signaling

14 Protein and Polypeptide Hormones: Synthesis and Release Figure 7-3: Peptide hormone synthesis, packaging, and release

15 DNA-binding domain in the receptor

16 Three classes of cell-surface receptors

17 Signaling pathway from a cell- surface receptor to the nucleus generates various kinds of intracellular signaling. A series of signaling proteins and small intracellular mediators relay the extracellular signal into the cell, causing a change in gene expression. The signal is amplified, altered (transduced), and distributed en route. The signaling pathway activates (or inactivates) target proteins that alter cell behaviour.

18 Intracellular signaling protein ac as molecular switches A signaling protein is activated by the addition of phosphate group and inactivated by the removal of phosphate.

19 Signal integration (A)Extracellular signals A and B both activate a different series of protein phosphosrylation, each of which leads to the phosphorylation of protein Y but at different sites on the protein. Protein Y is activated only when both of these sites are phosphorylated, and therefore it becomes active only when signals A and B are simulataneously present. (B)Extracellular signals A and B lead to the phosphorylation of two proteins, a and b, which then binds to each other to create the active protein.

20 Signaling through G-protein linked cell-surface receptors G-protein linked-receptors: Largest family of cell-surface receptors and found in all eukaryotes. Mediate the responses to an enormous diversity of signal molecules including hormones, neurotransmitters and local mediators. Signals molecule that activated them are varied in structure which includes proteins, peptides, derivatives of amino acids and fatty acids etc. Ligand binding causes a change in receptor comformation that activates a particular G-protein (abbrv. guanine-nucleotide binding protein). The structure consists of a single polypeptide chain that threads back and forth across the lipid bilayer seven times (aka. serpentine receptors). It is thought that G-protein linked-receptors that mediate cell-cell signaling in multicellular organisms evolved from sensory receptors that were possessed by their unicellular eucaryotic ancestors.

21 Structure of G-protein linked-receptors

22 Regulation of G-protein linked-receptors

23 The disassembly of an activated G-protein into two signaling components (A)In the unstimulated state, the receptor and G-protein are both inactive. (B)Binding of an extracellular signal to the receptor changes the conformation of the receptor, which in turn alters the conformation of the G-protein that is bound to the receptor. (C)The alteration of the α subunit of the G-protein allows it to exchange its GDP for GTP. This causes the G protein to break up into two active components – an α subunit and a βγ complex, both can regulated the activity of target proteins in the plasma membrane. The receptor stays active while the external signal molecule is bound to it, and can therefore catalyze the activation of many molecules of G protein.

24 The switching off of the G-protein α subunit by the hydrolysis of its bound GTP After a G-protein α subunit activates its target protein, it shuts itself off by hydrolyzing its bound GTP to GDP. This inactivates the α subunit, which dissociates from the target protein and reassociates with a βγ complex to re-form an inactive G protein.

25 Cyclic AMP is a second messenger for stimulatory G protein (G s ) In a reaction catalyzed by the enzyme adenylyl cyclase, cyclic AMP (cAMP) is synthesized from ATP through a cyclization that removes two phosphate groups as pyrophosphate. Cyclic AMP is unstable in the cell because it is itself hydrolyzed by a specific phosphodiesterase to form 5’-AMP.

26 The binding of an extracellular signal molecule to its G-protein linked- receptor leads to the activation of adenylyl cyclase and a rise in cyclic AMP concentration. The increase in cyclic AMP activates PKA in the cytosol and released catalytic subunits then move into the nucleus, where they phosphorylates cyclic AMP response element (CREB) gene regulatory element. Once phosphorylated, CREB recruits the coactivator CBP which stimulates gene transcription.

27 Inositol triphosphate and diacylglycrol as second messenger for G protein The activated receptor stimulates the plasma membrane-bound enzyme phospholipase C-β via G protein. It can be activated by the α subunit of G q or by βγ complex of another G protein, or by both. Two intracellular messenger molecules are produced when PI(4,5)P 2 is hydrolyzed by phospholipase C-β. Inositiol triphosphates diffuses through the cytosol and release Ca 2+ from the endoplasmic reticulum by binidng to and opening IP 3 -gated Ca 2+ release channels in the endoplasmic reticulum membrane. Diacylglycerol remains in the plasma membrane and together with phospatidylserine and Ca 2+, helps activate the enzyme protein kinase C which is recruited from the cytosol to the cytosolic face of the plasma membrane.

28 Three major families of trimetric G proteins Family members Action mediated by Functions IGsGs α Activates adenylyl cyclase; activates Ca 2+ channels G olf α Activates adenylyl cyclase in olfactory sensory neurons IIGiGi α Inhibits adenylyl cyclase βγ Activates K + channels GoGo βγ Activates K + channels; inactivates Ca 2+ channels α and βγ Activates phospholipase C-β GtGt α Activates cyclic GMP phosphoesterase in vertebrate photoreceptors IIIGqGq α Activates phospholipase C-β

29 Signaling through enzyme-linked cell-surface receptors Recognized through their role in responses to extracellular signal proteins that promote growth, proliferation, differentiation and survival of cells in animal tissues. Signal proteins are known as growth factors and usually act as loal mediators at every low conentration (about to M). Enzyme-linked receptors are transmembrane proteins (each subunit usually has only one) with their ligand-binding domain on the outer surface of the plasma membrane. Their cytosolic domain either has an intrinsic enzyme activity or associates directly with an enzyme. Six classes of enzyme-linked receptors: i) receptor tyrosine kinases ii) tyrosine-kinase-associated receptors iii) receptor-like tyrosine phosphatases iv) receptor serine/threonine kinases v) receptor guanylyl cyclases vi) histidine-kinase-associated receptors

30 Structure of receptor tyrosine kinase Consist of a single polypeptide chain with only one transmembrane segment. The extracellular portion of the receptor contains ligand-binding domain. The cytosolic portion of the receptor contains tyrosine residues that are in fact themselves targets for tyrosine kinase portion of the receptor. → receptor + protein kinase In some cases, the receptor and tyrosine kinase are two separate proteins. It can bind to the receptor and be activated when the receptor binds its ligand. → nonreceptor tyrosine kinase

31 Activation of receptor tyrosine kinase Signal transduction is initiated when ligand binds, causing the receptor tyrosine kinase to aggregate. → Two receptor molecules luster together within the plasma membrane when they bind ligand (dimerization) Tyrosine kinase associated with each receptor phosphorylates the tyrosine kinase of neighbouring receptors (autophosphorylation). Subsequently, relay proteins bind to the phosphorylated receptors and ativate subsequent protein to trigger a response.

32 RTK initiate a signal transduction cascade involving Ras and MAP kinase Upon ligand binding, RTK aggregate and undergo autophosphorylation. Once a receptor is phosphorylated at tyrosine residues in its cytosolic tails, proteins with SH2 domain such as GRB2 bind to the receptor, recruiting GEF for Ras. Ras-GEF causes the activation of Ras by helping it to release GDP and acquire GTP. Activated Ras then activates several downstream signaling pathways.

33 Activated Ras triggers the phosphorylation of protein kinase Raf. Activated Raf in turn phosphorylates serine and threonine residues in protein kinase MEK. MEK then phosphorylates threonines and tyrosine residues of mitogen-activated protein kinases (MAPKs). One of the funtion of MAPKs is to phosphorylate transcription factors that regulate gene expression.

34 kinase cascade – a series of protein kinases that phosphorylate each other in succession

35 Cross-talk of transduction pathway

36 Specificity of cell signaling The same ligand gives rise to different responses according to their cell type. → Cells differ in terms of their protein contents. → The same ligand gives rise to different responses: i) Cells differ in terms of their proteins ii) Different proteins respond differently to the same environmental signals (note, though, same receptors, different relay) iii) Different cells behave differently because some, but not all proteins can differ between cell types