Signal-Transduction Pathways

Transcription

1 Signal-Transduction Pathways Pal Bauer 2014/2015 Copyright 2007 by W. H. Freeman and Company

2 No men is an island entire of itself; every man Is a piece of the continent, a part of the main John Donne

3 Introduction Cells must respond adequately to external stimuli to survive. Cells respond to stimuli via cell signaling. Some signal molecules enter cells; others bind to cell-surface receptors.

4 Quorum sensing in bacteria

5 The slime mould Dictyostelium Discoideum

6 The Basic Elements of Cell Signaling Systems Extracellular messenger molecules transmit messages between cells.

7 In autocrine signaling, the cell has receptors on its surface that respond to the messenger Interleukine-1, FAS-L, TNF-alfa etc.

8 During paracrine signaling, messenger molecules travel short distances through extracellular space. Somatostatine, histamine, prostaglandins, quorum sensing etc.

9 During endocrine signaling, messenger molecules reach their target cells through the bloodstream.

10 Connexin gap junctions; ATP, GSH Anoikis, integrins, selectins, syndecans,cadherins etc.

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12 Chemical Synapse - Signal Transduction

13 Extracellular vesicles, exosomes, microvesicles

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15 Reception Binding of epinephrine to G protein-coupled receptor (1 molecule) Transduction Inactive G protein Active G protein (10 2 molecules) Inactive adenylyl cyclase Active adenylyl cyclase (10 2 ) ATP Cyclic AMP (10 4 ) Inactive protein kinase A Active protein kinase A (10 4 ) Inactive phosphorylase kinase Active phosphorylase kinase (10 5 ) Inactive glycogen phosphorylase Active glycogen phosphorylase (10 6 ) Response Glycogen Glucose 1-phosphate (10 8 molecules)

16 Direct Ligand Binding Plot and the Derived Scatchard Plot

17 Biological Activity and Receptor Occupancy 50% of maximum biological activity with ~18% of receptors occupied >80% of maximum biological activity with 50% of receptors occupied Epinephrine levels of ~10-10 M can stimulate glucogenolysis in liver cells, despite its relatively low binding affinity (K d ~ 10-5 M)

18 I. The Basic Elements of Cell Signaling Systems Receptors on or in target cells receive the message. Some cell surface receptors generate an intracellular second messenger through an enzyme called an effector. Other surface receptors recruit proteins to their intracellular domains.

19 Overview of signaling pathways

20 Signaling pathways consist of a series of proteins. Each protein in a pathway alters the conformation of the next protein. Protein conformation is usually altered by phosphorylation. Target proteins ultimately receive a message to alter cell activity. This overall process is called signal transduction.

21 A signal transduction pathway

22 A Survey of Extracellular Messengers and Their Receptors Extracellular messengers include: Small molecules such as amino acids and their derivatives (glutamate, acetylcholine, adrenaline, dopamine, TSH). Gases such as NO and CO Steroids Eicosanoids, which are lipids derived from fatty (arachidonic) acids. Various peptides and proteins

23 Receptor types include: G-protein coupled receptors (GPCRs) Receptor protein-tyrosine kinases (RTKs) Ligand gated channels Steroid hormone receptors Specific receptors such as B-and T-cell receptors

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25 I. G Protein-Coupled Receptors and Their Second Messengers

26 Signaling with G-Protein Coupled Receptors Receptors (GPCRs) integral membrane proteins with 7 transmembrane segments binding site for diverse ligands (hormones, odorants, tastants, light) >907 human GPCRs (384 olfactory receptors) G Proteins trimeric complexes of a, b and g subunits (20 Ga, 5 Gb, 12 Gg) Ga is GTPase switch protein (GDP off / GTP on) attached to membrane: Ga is acylated: Gg is prenylated Effectors adenylate cyclase, phospholipase C, phosphodiesterase, channels control levels of secondary messengers (camp, cgmp, DAG, IP 3 )

27 A GPCR and a G protein

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30 Lipid Rafts and Signal Transduction microdomains on surface of plasma membrane segregate proteins based on attached lipid acylated proteins in raft prenylated proteins not caveolin causes inward curvature forming caveolae localized in lipid rafts / caveolae: G-protein coupled receptors Tyr kinase receptors (some) not in lipid rafts: Ras and Gg subunit (prenylated)

31 Fluorescent Proteins Structure of green fluorescent protein (GFP) from jellyfish Chromophore is autocatalytically formed by cyclizing and oxidizing SYG sequence Site directed mutagenesis of GFP produced variety of other fluorescent proteins of different wavelengths

32 Assay for Measuring Protein Interactions Fluorescence Resonance Energy Transfer (FRET) uses emission of one chromophore as excitation for a second chromophore If proteins interact excitation of 1 st chromophore gives emission of 2 nd Applied to signal transduction study

33 Mechanism of receptormediated activation/inhibition by G proteins

34 G Protein-Coupled Receptors and Their Second Messengers Signal Transduction by G Protein-Coupled Receptors Ligand binding on the extracellular domain changes the intracellular domain. Affinity for G proteins increases, and the receptor binds a G protein intracellularly. GDP is exchanged for GTP on the G protein, activating the G protein. One ligand-bound receptor can activate many G proteins.

35 G Protein-Coupled Receptors and Their Second Messengers Termination of the Response Desensitization by blocking active receptors from turning on additional G proteins. G protein-coupled receptor kinase (GRK) modifies GPCR via phosphorylation. Proteins called arrestins compete with G proteins to bind GPCRs. Termination of the response is accelerated by regulators of G protein signaling (RGSs).

36 G Proteins and Effector Proteins Gas Gai Gaq G 12/13 activator camp inhibitor camp phosphoinositides unknown

37 Second Messengers *ER = endoplasmic reticulum; IP 3 = inositol 1,4,5-trisphosphate; PLC = phospholipase C; PI = phosphatidyl inositol; DAG = diacylglycerol; PLD = phospholipase D.

38 G Protein-Coupled Receptors and Their Second Messengers Second Messengers cyclic AMP The Discovery of Cyclic AMP It is a second messenger, which is released into the cytoplasm after binding of a ligand. Second messengers amplify the response to a single extracellular ligand.

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41 Formation of camp from ATP

42 Images of camp Transients in Cultured Aplysia Sensory Neurons. The cell was loaded with a fluorophore that would allow for the quantification of camp concentrations within the cell. A: Free camp in the resting cell is < 5 X 10-8 M. B: Stimulation with serotonin, activates adenylate cyclase increasing cytoplasmic camp to ~ 1 X 10-6 M (red), especially within fine processes with a high surface to volume ratio. Thus, within 20 sec of stimulation, the intracellular [camp] increased ~ 20-fold.

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44 The variety of processes that can be affected by changes in [camp]

45 G Protein-Coupled Receptors and Their Second Messengers Other Aspects of camp Signal Transduction Pathways Some PKA molecules phosphorylate nuclear proteins. Phosphorylated transcription factors regulate gene expression. Phosphatases halt the reaction cascade. camp is produced as long as the external stimulus is present.

46 Examples of hormone-induced responses mediated by camp

47 PKA-anchoring protein signaling

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49 G Protein-Coupled Receptors and Their Second Messengers Phosphatidylinositol-Derived Second Messengers Some phospholipids of cell membranes are converted into second messengers by activated phospholipases. Phosphatidylinositol Phosphorylation Phosphoinositides (PI) are derivatives of phosphatodylinositol.

50 Phosphatidylinositides in Signal Transduction Activation of Protein Kinase B PKB (Active) Ptdlns(4,3,5)P 3 (PIP 3 ) PI-3 Kinase Pathway PTEN ADP PI-3K ATP ADP ATP ADP Pi ATP Ptdlns PI-4K Ptdlns4P PIP-5K Ptdlns(4,5)P 2 PLC (PIP 2 ) IP 3 /DAG Pathway DAG Ins(1,4,5)P 3 (IP 3 ) Activation of Protein Kinase C PKC (Active) [Ca 2+ ]

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52 Cellular responses elicited by adding IP3

53 Phospholipid-based second messengers

54 G Protein-Coupled Receptors and Their Second Messengers Phosphatidylinositol-specific phospholipase C-b produces second messengers (IP 3 ) and diacylglycerol (DAG) derived from phosphatidylinositol-inositol triphosphate DAG activates protein kinase C, which phosphorylates serine and threonine residues on target proteins. The phosphorylated phosphoinositides form protein-binding domains, which are connected to the PH domains of participating proteins

55 IP 3 -Mediated Signal Transduction

56 Examples of responses mediated by Protein Kinase C

57 The Role of Calcium as an Intracellular Messenger Cytoplasmic calcium levels are determined by events within a membrane. Calcium levels are low in the cytosol (100 nm) because it is pumped out into the extracellular space and the membrane is highly impermeable to the ion. Calcium channels can be transiently opened by action potential or calcium itself (1 mm). Calcium binds to calcium-binding proteins (such as calmodulin), which affects other proteins.

58 Regulation of Cytosolic [Ca 2+ ] IP 3 -gated channels in ER release Ca 2+ into cytosol cytosolic [Ca 2+ ] lowers affinity of gated channels for IP 3 causes oscillation in cytosolic [Ca 2+ ] cytosolic [Ca 2+ ] measured using fluorescent Ca 2+ -binding dye Time course of cytosolic [Ca 2+ ] with α 1 -adrenergic receptor stimulation by epinephrine high sustained Ca 2+ release may be toxic

59 Experimental demonstration of localized release of intracellular Ca 2+

60 Calcium wave in a starfish egg

61 Calcium-induced calcium release

62 Examples of mammalian proteins activated by Ca 2+

63 Calmodulin

64 II: Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Protein-tyrosine kinases phosphorylate tyrosine residues on target proteins. Protein-tyrosine kinases regulate cell growth, division, differentiation, survival, and migration. Receptor protein-tyrosine kinases (RTKs) are cell surface receptors of the protein-tyrosine kinase family.

65 2) Receptor tyro sine kinase (RTK) family of receptors

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67 Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Receptor Dimerization Results from ligand binding. Protein kinase activity is activated. Tyrosine kinase phosphorylates another subunit of the receptor (autophosphorylation). RTKs phosphorylate tyrosines within phosphotyrosine motifs.

68 Activation of Protein Tyrosine Kinases Activation of a Tyr kinase by phosphorylation

69 Steps in the activation of RTK

70 Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Phosphotyrosine-Dependent Protein- Protein Interactions Phosphorylated tyrosines bind effector proteins that have SH2 domains and PTB domains. SH2 and PTB domain proteins include: Adaptor proteins that bind other proteins. Docking proteins that supply receptors with other tyrosine phosphorylation sites. Signaling enzymes (kinases) that lead to changes in cell. Transcription factors

71 A diversity of signaling proteins

72 Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction The Ras-MAP Kinase Pathway Ras is a G protein embedded in the membrane by a lipid group. Ras is active when bound to GTP and inactive when bound to GDP.

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74 The structure of a G protein and the G protein cycle GDI-guanine dissociation inhibitorinhibitor; GEF-guanine exchange factor; GAP- GTP-ase activator protein

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76 Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Ras-MAP kinase pathway Accessory proteins play a role: GTPase-activating proteins (GAPs) shorten the active time of Ras. Guanine nucleotide-exchange factors (GEFs) stimulate the exchange of GDP for GTP. Guanine nucleotide-dissociation inhibitors (GDIs) inhibit release of GDP.

77 Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Ras-MAP kinase pathway (continued) The Ras-MAP kinase cascade is a cascade of enzymes resulting in activation of transcription factors. Adapting the MAP kinase to transmit different types of information: End result differs in different cells/situations. Specificity of the MAP kinase response due to differences in the types of kinases participating and differences in spatial organization of components.

78 The steps of a generalized MAP kinase cascade

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80 Mammalian Ras activation SH2 Domain PI3K P P GRB2 (DRK) SOS GAP P P PLC Ras GTP Downstream pathways

81 Ca 2+ signaling can be activated by RTKs via PLC g PI3K P P GRB2 GAP P P PLC Ca 2+ signaling

82 RTKs can activate PI3-Kinase PI3K P P GRB2 GAP P P PLC Other signaling pathways Cell survival

83 PI3K P P GRB2 Src P P PLC Cell proliferation, Gene expression,

84 Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction. Signaling by the Insulin Receptor Insulin regulates blood glucose levels by increasing cellular uptake of glucose. The insulin receptor is a protein-tyrosine kinase Autophosphorylated receptor associates with insulin receptor substrate proteins (IRSs). IRSs bind proteins with SH2 domains, which activate downstream signal molecules. SH2 domain-containing proteins are kinases that phosphorylate a lipid, PI 3-kinase (PI3K).

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86 The role of tyrosine-phosphorylated IRS in activating a variety of signaling pathways

87 Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Glucose Transport PKB regulates glucose uptake by GLUT4 transporters. GLUT4 transporters reside in intracellular membrane vesicles. Vesicles fuse with the membrane in response to ligand binding to the IR. Diabetes mellitus is caused by defects in insulin signaling and Type 2 diabetes is caused by gradual insensitivity to insulin.

88 Regulation of glucose uptake in muscle and fat cells by insulin

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90 Convergence, Divergence and Crosstalk Among Different Signaling Pathways Signaling pathways can converge, diverge, and crosstalk as follows: Signals form unrelated receptors can converge to activate a common effector. Identical signals can diverge to activate a variety of effectors. Signals can be passed back and forth between pathways as a result of crosstalk.

91 Examples of convergence, divergence, and crosstalk among signal transduction pathways

92 Convergence, Divergence and Crosstalk Among Different Signaling Pathways Convergence GPCRs, receptor tyrosine kinases, and integrins bind to different ligands but they all can lead to a docking site for Gbr2.

93 Convergence of signals transmitted from a GPCR, an integrin, and receptor tyrosine kinase

94 Convergence, Divergence and Crosstalk Among Different Signaling Pathways (3) Divergence all of the examples of signal transduction so far are evidence of divergence of how a single stimulus sends signals along a variety of different pathways.

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96 Convergence, Divergence and Crosstalk Among Different Signaling Pathways (4) Crosstalk more and more crosstalk is found between signaling pathways: camp can block signals transmitted through the MAP kinase cascade. Ca 2+ and camp can influence each other s pathways.

97 An example of crosstalk between two major signaling pathways

98 Intracellular Signaling Pathways activated by RTKs and GPCRs

99 Two Stages of Amplification Adenylyl cyclase activity is modulated by the interplay of stimulatory and inhibitory G proteins. Hormone binding to β 1 - and α2-receptors activates adenylyl cyclase, whereas hormone binding to α 2 - receptors leads to inhibition of adenylyl cyclase.

100 The Role of NO as an Intracellular Pathway Nitric oxide (NO) is both an extracellular and intercellular messenger with a variety of functions. NO is produced by nitric oxide synthase. NO stimulates guanylyl cyclase, making cgmp. cgmp decreases cytosolic calcium and relaxes smooth muscle. NO also plays a role in male arousal.

101 Signal transduction by means of NO and cgmp

102 Steroid Hormones: Features Cholesterol-derived Lipophilic and can enter target cell Cytoplasmic or nuclear receptors (mostly) Activate DNA for protein synthesis Slower acting, longer half-life Examples Cortisol, estrogen, and testosterone

103 Steroid Hormones: Structure Cholesterol is the parent compound for all steroid hormones. modified by enzymes to make steroid hormones such as In adrenal cortex In ovary Ovary Adrenal cortex Aldosterone Cortisol Estradiol (an estrogen) Figure 7-6

104 Steroid Hormones: Action Blood vessel Protein carrier 1 Steroid hormone 2a 2 Cell surface receptor Rapid responses Nucleus 1 2 Most hydrophobic steroids are bound to plasma protein carriers. Only unbound hormones can diffuse into the target cell. Steroid hormone receptors are in the cytoplasm or nucleus. Cytoplasmic receptor Nuclear receptor DNA 2a Some steroid hormones also bind to membrane receptors that use second messenger systems to create rapid cellular responses. Interstitial fluid Cell membrane Endoplasmic reticulum New proteins 5 4 Translation 3 Transcription produces mrna The receptor-hormone complex binds to DNA and activates or represses one or more genes. Activated genes create new mrna that moves back to the cytoplasm. Translation produces new proteins for cell processes. Figure 7-7

105 Steroid Receptor Structure This superfamily of ligand-activated transcription factors is also structurally related. Three well conserved regions: -Hormone binding domains (HBD) in carboxyl terminus -DNA-binding domain (DBD) 5 to ligand binding domain A nonconserved hypervariable region, which may contribute to transcriptional activity of receptor hypervariable DBD HBD

106 Not All Intracellular Receptors are Associated with HSPs. HSPs bind to glucocorticoid, mineralocorticoid, androgen, progesterone, and estrogen receptors in absence of hormone. However, receptors for thyroid hormone, retinoic acid, and vitamin D are not bound to HSPs. This second group of receptors is bound to their hormone response element (HRE) on 5 flanking region of target genes, but are inactive until hormone binds to them.

107 Steroid Receptors bind to Hormone Response Elements (HREs) on DNA Following hormone binding, intracellular receptors act as transcription factors, binding to hormone response elements (HREs) on the 5 flanking region of target genes. HRE 5 flanking region target gene

108 Steroid Receptors bind to Hormone Response Elements (HREs) on DNA Hormone Progesterone, Androgen, Glucocorticoid, Mineralocorticoid Consensus HREs AGAACAnnnTGTTCT Estrogen Thyroid hormone Retinoids Vitamin D AGGTCAnnnTGACCT AGGTCATGACCT

109 Palindromic Sequences Allow Binding of Receptors as Dimers 5 -AGAACAnnnTGTTCT- 3 H NNN A T C G A T A T G C A T H Transcription TATA EXON 1...

110 Ligand gated ion channels Gated by ligands present outside of the cell In fact they are receptors All of them are nonselective cation channels Mediate effects of neurotransmitters

111 GPCRs that Regulate Ion Channels: Muscarinic Acetylcholine Receptor The neurotransmitter, acetylcholine (ACH) binds to two types of receptors known as the nicotinic and muscarinic acetylcholine receptors. The nicotinic receptor is itself a ligandgated ion channel that opens on ACH binding. This receptor is located in the neuromuscular junctions of striated muscle. The muscarinic ACH receptor, is a GPCR found in cardiac muscle cells that is coupled to an inhibitory G protein The binding of ACH to this receptor triggers dissociation of G ai - GTP from G ßg, which in this case, directly binds to and opens a K + channel. The movement of K + down its concentration gradient to the outside of the cell, increases the positive charge outside the membrane, hyperpolarizing the cell. This results in the slowing of heart rate.

112 Acetylcholine Receptor g (or e) ACh a b consists of a pentamer of protein subunits, with two binding sites for acetylcholine, which, when bound, alter the receptor's configuration and cause an internal pore to open. ACh d This pore allows Na+ ions to flow down their electrochemical gradient into the cell.

113 Nicotinic Acetylcholine Receptor A ligand gated ion channel

114 the resting (closed) ion channel to acetylcholine (ACh) produces the excited (open) state. Longer exposure leads to desensitization and channel closure. Acetylcholine binding sites ACh Na +, Ca 2+ Continued excitation Outside Inside Resting (gate closed) Excited (gate open) Desensitized (gate closed) ACh