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SIGNAL TRANSDUCTION A. OVERVIEW OF RECEPTORS AND SIGNALLING RECEPTOR FAMILIES MECHANISMS OF SIGNALLING BY RECEPTORS AMPLIFICATION OF SIGNALS KEY FUNCTION.

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Presentation on theme: "SIGNAL TRANSDUCTION A. OVERVIEW OF RECEPTORS AND SIGNALLING RECEPTOR FAMILIES MECHANISMS OF SIGNALLING BY RECEPTORS AMPLIFICATION OF SIGNALS KEY FUNCTION."— Presentation transcript:

1 SIGNAL TRANSDUCTION A. OVERVIEW OF RECEPTORS AND SIGNALLING RECEPTOR FAMILIES MECHANISMS OF SIGNALLING BY RECEPTORS AMPLIFICATION OF SIGNALS KEY FUNCTION OF PHOSPHORYLATION PROTEIN INTERACTION DOMAINS

2 Objectives of this lecture Recognize the different types of receptors and the mechanisms they use to signal into cells Understand the importance of signal amplification Understand the basic mechanisms of protein phosphorylation and the type of kinases Identify some of the key protein interaction domains that function in signalling pathways Be aware of applicability of these studies to virtually all disease processes (cancer is highlighted)

3 A Cascade of Signals from Membrane to Nucleus Downward, Nature 2001

4 CYTOKINE PRODUCTION / HORMONE ACTION Inducing Stimulus Cytokine- producing cell Target Gene Activation Biological Effect Target cell Receptor PARACRINE Distant Cell Circulation Nearby Cell ENDOCRINE AUTOCRINE Cytokine gene

5 CLASSIFICATION OF RECEPTORS IN FAMILIES A.Receptors for Growth and Differentiation Factors - have associated enzyme activity B. Serpentine Receptors - coupled to G proteins C. Intracellular Receptors - bind hormone and act as transcription factors D. Channel Forming Receptors - receptors for neurotransmitters E. Immune System Receptors - T cell, B cell, Ig receptors

6 A. Receptors for Growth and Differentiation Factors In general, tyrosine kinase activity is involved in receptor signalling (some serine/threonine kinase receptors, some guanylate cyclase-encoding) Receptors with intrinsic tyrosine kinase domain: - EGF, PDGF, FGF, SLF/c-kit have single subunits - insulin, IGF-I have multiple subunits, 2 2 - hepatocyte growth factor (c-met receptor),

7 Growth Factor Receptors with Tyrosine Kinase Domains Share Common Structural Features

8 8 Figure 1 Receptor Tyrosine Kinase Families Human receptor tyrosine kinases (RTKs) contain 20 subfamilies, shown here schematically with the family members listed beneath each receptor. Structural domains in the extracellular regions, identified... Lemmon & Schlessinger, Cell 2010

9 DIMERIZATION is a Key Concept In Understanding The Signalling Events Transmitted By Growth Factor Receptors Schlessinger, Cell 2000

10 10 Lemmon & Schlessinger, Cell 2010 Models depicting various means by which extracellular domains allow for dimerization

11 11 Models of intracellular domain kinase activation Lemmon & Schlessinger, Cell 2010

12 Receptors having associated tyrosine kinase Hemopoietin receptor family - includes receptors for interleukins and colony stimulating factors -primarily found in hematopoietic cells intracellular associated tyrosine kinases (JAKs) activated by ligand binding to receptor

13 IL-3, IL-5 and GM-CSF are Examples of Hemopoietin Receptors That Share a Common Subunit

14 B. Serpentine receptors, or G-protein coupled receptors (GPCRs) 7 transmembrane domains; extracellular domains are responsible for creating a ligand binding site eg. epinephrine, muscarinic acetylcholine receptor, rhodopsin coupled to G proteins via intracellular portion of receptor Signalling: via G protein transducers bind GTP in active state; hydrolyzed to GDP when inactive amplification of signals

15 STRUCTURE OF A TYPICAL SERPENTINE RECEPTOR

16 C. Intracellular Receptors translocated from cytosol to nucleus when bound with ligand; DNA binding and transcription activation domains Signalling: direct binding to DNA, in the presence of ligand, to activate transcription

17 Intracellular Receptors for Various Hormones have Conserved Structural Features Inhibitory protein complex Hormone binding site DNA binding domain Hormone DNA binding site exposed N C N CN CN C N C NC DNA binding domain Cortisol R Estrogen R Progesterone R Vitamin D R Thyroid hormone R Retinoic Acid R Transcription activation domain

18 D. Channel Forming Receptors in neural and muscle tissue eg.acetylcholine, dopamine, glycine, -aminobutyrate (GABA) structures with 4 or 5 subunits that each have several transmembrane domains; subunits cluster to form a gated channel Signalling: function at nerve and muscle synapses to propagate an electrical signal by transport of ions

19 E. Immune system receptors B and T cell receptors; consist of multiple subunits associated tyrosine kinases activated to phosphorylate ITAMs – Immune receptor Tyrosine- based Activation Motifs ITAM's serve as docking sites for other tyrosine kinases that are activated and subsequently activate signalling pathways that involve a series of intermediate tyrosine phosphorylated adaptor proteins. Signalling pathways utilized are similar to those of growth factor receptor tyrosine kinases.

20 The T-Cell Receptor Recognizes Antigens Bound to MHC on Antigen Presenting Cells

21 Signal transduction is the means by which molecular responses are propagated within a cell A cell senses its environment by way of signalling molecules (starting with receptors) and the resulting changes in molecular shapes or activities cause corresponding changes in cell behaviour Signal transduction studies aim to explain (molecularly) all aspects of the behaviour of an individual cell, from its growth and division, to its differentiation into a more specialized cell type, and its death by apoptosis. B. MECHANISMS OF SIGNAL TRANSDUCTION

22 G Protein Coupling to Serpentine Receptor Results in GTP/GDP Exchange and Dissociation of the Active G protein Subunit

23 Signalling Events Result in Enormous Amplification of Downstream Messengers to Affect Many Targets

24 PHOSPHORYLATION Protein kinases catalyze the transfer of the g- phosphate from nucleotide triphosphate (usually ATP) to a hydroxyl acceptor site on a protein Kinase Protein + NTPProtein-P + NDP Phosphatase Serine/Threonine Kinases; eg. cAMP-dependent protein kinase, Protein kinase C; MAP kinases Tyrosine Kinases; eg. EGF, PDGF, Insulin receptors, src family of oncogenes, JAK family Dual specificity kinases; eg. MEKs

25 25 Protein Phosphorylation Network

26 N-H H-C-CH2-OH O=C N-H H-C-CH2-O-P-O - O=C O O-O- Serine Phospho-Serine Phosphorylation causes a dramatic change in charge on a protein

27 Function of Protein Phosphorylation Addition of a highly charged phosphate group alters a protein's surface charge and its structure Numerous mechanisms by which phosphorylation can alter the function of a protein (switches) Tyrosine Phosphorylation - Results in formation of new sites of protein-protein interaction, mediated by SH2 or PTB domains Ser/Thr Phosphorylation, acts primarily to modulate activity of protein/enzyme that gets phosphorylated, but may also result in altered protein binding

28 Phosphorylation in Signal Transduction Pawson & Scott, TIBS, 2005

29 Other Kinases Lipid Kinases; e.g. PI Kinases can phosphorylate various positions on the inositol ring of the lipid Phosphatidylinositol Phosphorylation of sugars, nucleotides and many other small molecules, all mediated by kinases (these are usually involved in metabolic pathways as opposed to signalling pathways)

30 TURN-OFF SIGNALS For every signal transmitted into cells there must be a means of regulating, or turning off, the signal In the case of phosphorylation, phosphatases play a key role in reversing the reaction. Phosphatases include tyrosine, serine/threonine and lipid phosphatases. In the case of G proteins, reversal is by breaking down the guanine nucleotide by GTPase activity Degradation of ligand or its dissociation from receptor stops signalling at the receptor, although downstream events may still proceed

31 Protein Interaction Domains Many signalling pathways proceed via protein-protein interaction events Several domains identified that serve as 'cassettes' of protein 3D structure. In some cases, sequence homology is very weak, yet similar 3D structures have been demonstrated. The primary functions are to alter activity of an enzyme, or to change the location of an enzyme so it is placed close to its substrate (e.g. enzymes acting on lipids translocated to the plasma membrane)

32 Protein Interaction Domains Function of some domains depends on phosphorylation state; e.g. SH2 binding to phosphotyrosine Some show constitutive binding; e.g. SH3 to poly-proline motifs Others may bind specific second messengers to alter function of a protein, or its location in the cell; PH domains binding to lipids

33 Protein Modules and Docking Proteins Schlessinger, Cell 2000

34 SH Domains Src Homology - first noticed by comparison of src (the first oncogene) sequence with other proteins SH1 - tyrosine kinase domain SH2 - binds specific phosphotyrosines, with hydrophobic a.a.'s on C-term side of PY SH3 - binds polyproline motifs; binding constitutive PTB Domains Phosphotyrosine Binding Domains Bind phosphotyrosine in a binding pocket, like SH2, but specificity is determined by residues on N-term. side of PY

35 Pleckstrin Homology (PH) Domains First identified in Pleckstrin, a major protein kinase substrate first identified in platelets Shown to mediate protein interactions in a few cases, but primarily protein binding to lipids (mainly various forms of phosphorylated phosphatidylinositols)

36 Ras (GDP) P GRB2 (adaptor with both SH2 and SH3) SH2 binds to P-Y SOS Guanine nucleotide exchange factor; Bound to SH3 of GRB2 Via poly-proline Ras (GTP) raf MEKerk1/2 Transcription Factors Phosphorylation (threonine/tyrosine) Phosphorylation GTP exhanges with GDP Association via ras binding domain p21ras to erk - a key signalling pathway OUT IN

37 37 A Genetic vs Molecular Description of the Ras Pathway

38 38 Extracellular Signal TKR P85/p110 PI3K 4,53,4,53,4 5Pase PDK1 PKB 308 473 ?PDK2 NUCLEUS CYTOPLASM The Basics of PI 3-kinase Signalling PI 3-kinase phosphorylates PI(4,5)P2 in the plasma membrane

39 39 Control of Cell survival by PI3K/PKB Duronio, Biochem J, 2008

40 Many Signalling Proteins May Act as Oncogenes When Mutated or Overexpressed Nuclear proteins Myc fos jun PDGF EGF Growth Factors M-CSF Membrane-associated Tyrosine kinases src ras proteins GTP-binding proteins PDGF Receptor EGF Receptor (erb B) M-CSF Receptor (fms) Tyrosine kinase Receptors Cytoplasmic Tyrosine kinases (fps/fes) (raf) Cytoplasmic Ser/Thr kinases Thyroid Hormone Receptor (erb B) Steroid-type Growth factor Receptors

41 REFERENCES – Signal transduction (Duronio, lecture 1) The Ins and Outs of Signalling. J. Downward. Nature 411: 759 - 762 (2001). Kinome signaling through regulated protein-protein interactions in normal and cancer cells. T. Pawson and M. Kofler. Curr Opinion Cell Biol 21: 147-53 (2009). Protein phosphorylation in signaling - 50 years and counting. T. Pawson and J. Scott. TIBS 30: 286-290 (2005). Cell signaling by receptor tyrosine kinases. J. Schlessinger. Cell 103: 211 - 225 (2000). Cell signaling by receptor tyrosine kinases. M.A. Lemmon and J. Schlessinger. Cell 141: 1117- 1134 (2010). V. Duronio, The Life of a Cell – Apoptosis Regulation by the PI3K/PKB Pathway. Biochem J. 415: 333-344 (2008). For further in depth study: STKE, Science website http://stke.sciencemag.org/ http://stke.sciencemag.org

42 Mechanisms of Signalling by RTKs A. PKB Activation by Phosphorylation B. PI3K activation by pY binding and localization to plasma membrane C. PLCg activation by pY binding, phosphorylation and localization to membrane

43 Multiple Effectors Regulated by RTKs


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