Presentation on theme: "A. OVERVIEW OF RECEPTORS AND SIGNALLING"— Presentation transcript:
1A. OVERVIEW OF RECEPTORS AND SIGNALLING SIGNAL TRANSDUCTIONA. OVERVIEW OF RECEPTORS AND SIGNALLINGRECEPTOR FAMILIESMECHANISMS OF SIGNALLING BY RECEPTORSAMPLIFICATION OF SIGNALSKEY FUNCTION OF PHOSPHORYLATIONPROTEIN INTERACTION DOMAINS
2Objectives of this lecture Recognize the different types of receptors and the mechanisms they use to signal into cellsUnderstand the importance of signal amplificationUnderstand the basic mechanisms of protein phosphorylation and the type of kinasesIdentify some of the key protein interaction domains that function in signalling pathwaysBe aware of applicability of these studies to virtually all disease processes (cancer is highlighted)
3A Cascade of Signals from Membrane to Nucleus A schematic representation of how activation of a cell surface receptor by ligand binding can activate a cascade of events, first in the cytoplasm and most often regulating gene expression.Downward, Nature 2001
4CYTOKINE PRODUCTION / HORMONE ACTION Inducing StimulusCytokine-producing cellAUTOCRINECytokinegeneNearby CellReceptorPARACRINEA textbook representation of hormone action. The mediators can be called hormones as a general term, but alternatively, we could be talking about growth factors, cytokines (usually reserved for growth factors that affect cells of the blood (or hematopoietic) system), chemokines (which regulate cell movement by chemotaxis), hormones (which generally regulate cellular function or metabolism), or other small molecule mediators, including neurotransmitters.TargetGene ActivationTarget cellCirculationDistant CellENDOCRINEBiological Effect
5CLASSIFICATION OF RECEPTORS IN FAMILIES Receptors for Growth and Differentiation Factors- have associated enzyme activityB. Serpentine Receptors- coupled to G proteinsC. Intracellular Receptors- bind hormone and act as transcription factorsD. Channel Forming Receptors- receptors for neurotransmittersE. Immune System Receptors- T cell, B cell, Ig receptorsThis is a grouping of the receptors that I have used, that includes just about all types of receptors. There are both structural and functional features that are common to each.In some cases, the types of agents that act on the receptors can be very similar, while in other cases there can be a wide diversity of ligands. (Ligand is a general term used to describe any agent that binds with high specificity to a receptor.)
6A. 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, α2β2- hepatocyte growth factor (c-met receptor), αβFor this class, the major functional feature is the tyrosine kinase activity - this can be either intrinsic to the receptor, or be activated by protein-protein associations. However, this class of receptors also includes some that utilize other enzymatic activities for signalling. The receptor proteins span the plasma membrane once, but may consist of multiple subunits. In general, they promote cell division, but also can modulate cell growth to promote differentiation. This is usually in the context of other signals that are received by the cell at various stages of development.
7Growth Factor Receptors with Tyrosine Kinase Domains Share Common StructuralFeaturesThese simple representations indicate domains that are common to the different classes of TK receptors. The black box indicates a conserved tyrosine kinase domain, which can be split into two sections, with a kinase insert domain (KI). In classes I and II, the open box represents cysteine-rich domains that form the 3D structure of the receptor at the cell surface. The loops in types III and IV are known as immunoglobulin(Ig)-like domains. In both of these domains, there is relatively little homology, but key residues that determine structure are highly conserved.
8Figure 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
9By Growth Factor Receptors DIMERIZATIONis a Key ConceptIn UnderstandingThe SignallingEvents TransmittedBy Growth Factor ReceptorsWhether the receptors consist of only one type of subunit, or two or more different subunits, a common concept is the subunit associations that result in either dimers or multimers. The association may be induced by addition of the receptor’s ligand, or pre-formed dimers can have their conformation altered or stabilized by addition of ligand. The latter is supported by studies showing that inhibitors of phosphatases can activate signalling in the absence of ligand - i.e. by preventing the counteracting dephosphorylation, tyrosine kinase inhibitors allow accumulation of phosphorylated receptors.Schlessinger,Cell 2000
10Models depicting various means by which extracellular domains allow for dimerization Protein structure studies have revealed various mechanisms by which ligands (in red) can mediate dimerization although one extreme is in ErbB where the receptor is pre-dimerizedLemmon & Schlessinger,Cell 2010
11Models of intracellular domain kinase activation Lemmon & Schlessinger,Cell 2010
12Receptors having associated tyrosine kinase Hemopoietin receptor family - includes receptors for interleukins and colony stimulating factorsprimarily found in hematopoietic cellsintracellular associated tyrosine kinases (JAK’s) activated by ligand binding to receptorAs opposed to receptor tyrosine kinases (RTK’s) in which the enzyme is part of the receptor, many receptors have no intrinsic enzymatic activity and instead are associated with a separate tyrosine kinase, which is not considered part of the receptor. These tyrosine kinases are activated by receptor dimerization or oligomerization that is induced by ligands, thus serving the same role as the intrinsic activity is RTK’s. The associated TK can phosphorylate the receptors themselves, or intermediate adaptor proteins that serve to bind signalling molecules.
13IL-3, IL-5 and GM-CSF are Examples of Hemopoietin Receptors That Share a Common SubunitThe IL-3, IL-5 and GM-CSF sub-group of hemopoietin receptors share a common beta subunit that is primarily responsible for signal transduction (activation of JAK’s). The alpha subunit provdes the high affinity ligand binding site, although the highest affinity is obtained by binding of the cytokine to the two subunits together There is a unique situation in the mouse system - while there is a common beta subunit used by each of the receptors, there is a closely related beta subunit that is only utilized in the IL-3 receptor). This theme of subunit sharing occurs with other groups of cytokines as well. For example, IL-2, 4 and 13 share a common gamma subunit (these receptors have 3 subunits); IL-6, OSM, CNTF, LIF receptors share a common subunit called gp130.
14B. Serpentine receptors, or G-protein coupled receptors (GPCR’s) 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 receptorSignalling: via G protein transducersbind GTP in active state; hydrolyzed to GDP when inactive amplification of signalsThis is probably the largest single group of receptors, with hundreds of types. There are a large variety of ligands that bind to these receptors, ranging from proteins such as chemokines to small molecules such as epinephrine and acetylcholine. They are also called G-protein-coupled receptors based on their signalling function, or 7- spanners which describes their structure. The serpentine description comes from the snake-like manner in which the polypeptide crosses the membrane using 7 transmembrane domains.
15STRUCTURE OF A TYPICAL SERPENTINE RECEPTOR This schematic shows the structure of a serpentine receptor, with each circle representing an amino acid (aa). The shaded aa’s represent the predominantly hydrophobic amino acids of the transmembrane (TM) domains (embedded in the lipid bilayer). As illustrated here, a large proportion of the structure consists of the TM, as opposed to the receptors with a single TM; those may have hundreds of aa’s in each of the extracellular and intracellular domains, with only the aa’s that make up the TM. The extracellular loops of the serpentine receptor come together to form the ligand binding site, and the loops of the intracellular domain interact with signalling molecules known as G proteins.
16C. Intracellular Receptors translocated from cytosol to nucleus when bound with ligand; DNA binding and transcription activation domainsSignalling: direct binding to DNA, in the presence of ligand, to activate transcriptionThese receptors include a large family of steroid and non-steroid hormone receptors, as well as many receptors still classified as ‘orphan’ receptors since the natural ligand has not been characterized. Association of ligand with receptor results in a conformational change that exposes a nuclear localization signal (NLS). This most often results from the receptor in the inactive state being bound to a heat shock protein, and binding of hormone causing release of the hsp and unmasking of the NLS.
17Intracellular Receptors for Various Hormones have Conserved Structural FeaturesDNA bindingdomainTranscriptionactivation domainInhibitoryprotein complexNCCortisol RHormonebinding siteNCEstrogen RNCProgesterone RDNA binding domainNCVitamin D RHormoneIn this schematic, the binding of hormone is shown to cause release of the inhibitory protein (hsp), which also serves to expose a DNA binding site. The DNA binding domain is a conserved (but not identical) sequence. The hormone binding site at the C-terminus and the transcription-activating domain at the N-terminus are unique to each specific receptor, and will determine the specific site of the DNA to which the receptor will bind and thus turn on gene transcription.NCThyroid hormone RDNA bindingsite exposedNCRetinoic Acid R
18D. Channel Forming Receptors in neural and muscle tissue eg.acetylcholine, dopamine, glycine, γ-aminobutyrate (GABA) structures with 4 or 5 subunits that each have several transmembrane domains; subunits cluster to form a gated channelSignalling: function at nerve and muscle synapses to propagate an electrical signal by transport of ionsThese are highly specialized receptors primarily in neural and muscle cells, which form an ion channel made up of several interacting subunits. The channel allows regulated movement of ions in response to neurotransmitters.
19E. Immune system receptors B and T cell receptors; consist of multiple subunits associated tyrosine kinases activated to phosphorylate ITAM’s – 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.These are speciallized receptors of the immune system. Besides the T and B cell receptor (each a single class, but there are millions of individual structures that determine specific antigen binding), there are also immunoglobulin receptors such as IgE receptors on mast cells, critical in allergic responses.
20The T-Cell Receptor Recognizes Antigens Bound to MHC on Antigen Presenting CellsAntigens are created within an antigen presenting cells by degradation of larger molecules, and the resulting smaller peptides are bound in a groove of the MHC molecule. The T cell receptor (TCR) recognizes the antigen via the alpha and beta subunits (analogous to an immunoglobulin structure). At the same time, there are other molecules such as CD4 that must recognize the MHC molecule. The CD4 has an associated tyrosine kinase that when in proximity with the TCR
21B. MECHANISMS OF SIGNAL TRANSDUCTION Signal transduction is the means by which molecularresponses are propagated within a cellA cell senses its environment by way of signallingmolecules (starting with receptors) and the resulting changes in molecular shapes or activities cause corresponding changes in cell behaviourSignal 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.These are a few descriptors regarding concepts in cell signalling
22G Protein Coupling to Serpentine Receptor Results in GTP/GDP Exchange and Dissociation of the ‘Active’ G protein SubunitFrom ‘Molecular Biology of the Cell’; a cartoon that depicts the multiple steps involved in G protein signalling. Serpentine receptors interact with G proteins that consist of 3 subunits (αλπηα,βετα,γαμμα). The alpha subunit binds GDP when inactive or GTP when activated as a result of receptor assocation. Concurrently, the alpha subunit dissociates from beta/gamma, which can also have its own signalling targets. The alpha subunit can then interact with a target signalling molecule, such as adenylate cyclase, which is the enzyme that produces cAMP from ATP. When the receptor is no longer activating the alpha subunit, the GTP is hydrolyzed to GDP and the alpha subunit returns to the inactive state.
23Signalling Events Result in Enormous Amplification of Downstream ‘Messengers’to Affect Many TargetsAmplification of signalling may be one reason for there being multiple steps in a signal transduction pathway. Again looking at the G proteins coupling to cAMP production, a single receptor when activated by a single ligand molecule can interact with several G proteins, each of which becomes transiently activated to activate an adenylate cyclase (AC) enzyme. Each of the activated AC’s can produce many cAMP molecules. In principle, each cAMP can activate a molecule of protein kinase A( PKA; one of its primary targets). Since PKA is also an enzyme, it can catalyze the phosphorylation of many targets. If the target is itself an enzyme, then each of those enzymes can also produce many product molecules. Thus from a single ligand/receptor interaction, there may be thousands of individual molecules that are affected.
24Protein + NTP Protein-P + NDP Phosphatase PHOSPHORYLATION Protein kinases catalyze the transfer of the g- phosphate from nucleotide triphosphate (usually ATP) to a hydroxyl acceptor site on a proteinKinaseProtein + NTP Protein-P + NDPPhosphatase 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. MEK’sDefinitions surrounding phosphorylation. Kinases are the key enzymes that catalyze the reactions, and they are classified according to the specific amino acid being phosphorylated. Changes in the protein target adds a highly negatively charged residue at a site where there was previously a polar amino acid, and thus there are many potential ways this can affect protein structure or activity.
25Protein Phosphorylation Network This network of phosphorylation events was published in Linding et al Cell, 29, (2007). The outside edges where lines make contact represent kinases or substrates
26Phosphorylation causes a dramatic change in charge on a protein N-HN-HO-H-C-CH2-OHH-C-CH2-O-P-O-OO=CO=CThe addition of a phosphate group on a serine residue replaces a polar hydroxyl group with a phosphate group having 2 negative charges at physiological pH.SerinePhospho-Serine
27Function 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 bindingprotein (or lipid) phosphorylation can have dramatic effects that directly alter the activity of the target, or alter its structure so that it has new functions or makes new interactions based on the presence of the additional phosphate group(s)
28Phosphorylation in Signal Transduction Transfer of phosphates act as a key type of signal in many types of signal transduction events.Pawson & Scott,TIBS, 2005
29Other 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)PI 3-kinase is a very important signalling enzyme, but there are numerous other PI kinases that phosphorylate other sites on the inositol ring. There are also several forms of PI 3-kinase, some which preferentially phosphorylate PI itself, vs ones that prefer PI-4,5-P2, for example, to yield PI-3,4,5-P3
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 proceedWithout a means of turning off a positive signal from a receptor, responses to a signal become uncontrolled. A good example of this is when a receptor activates the PI 3- kinase pathway, which leads to phosphorylation of the lipid phosphatidylinositol at the 3 position. The phosphatase that reverses that reaction is called PTEN. Mutations in PTEN (which normally acts as a ‘tumour suppressor’) are among the most commonly occurring mutations in human cancers. Besides reversal of phosphorylation, or breakdown of GTP bound to a G protein, the regulation at the level of the receptor may be by the removal of the positively acting ligand.
31Protein 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)A key defining feature of many signal transduction processes is the interactions made by the signalling proteins affected within the pathway. Since each of the individual components may interact with more than one partner, there are possibilities of a network of signals being propagated, as opposed to a linear pathway. The domains of protein structure that mediate the interactions can function in many different contexts. Experimentally, these domains can be taken out of their original protein sequence and fused into another context to test for the activity of the domain (domain swapping experiments). This has yielded much information about the normal functions.
32Protein 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
33Protein Modules and Docking Proteins Signalling proteins are recruited to tyrosine kinase-encoding receptors or to docking proteins (B) via SH2 and PTB domains. The signalling proteins may contain one or more other localization domains, including PH (PI lipid binding) SH# or WW domains that interact with poly-prolines, PDZ that bind to hydrophobic C-termini, or FYVE domains that bind PI3P. The associated enzyme activity of the signalling proteins may be altered by the binding of the other domains, or simply by being in proximity with substrates. Most adaptor proteins contain multiple phosphotyrosine sites that act as docking sites for SH2 and PTB domain containing proteins.Schlessinger,Cell 2000
34SH 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 constitutivePTB Domains Phosphotyrosine Binding Domains Bind phosphotyrosine in a binding pocket, like SH2, but specificity is determined by residues on N-term. side of PYThe ‘SH’ domains can be considered a UBC discovery, as Tony Pawson was working in the Microbiology Dept. and found similarities in sequence between src family oncogenes and the viral fps/fes oncogenes that he was working on.
35Pleckstrin 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)Although PH domain nomenclature came from the protein pleckstrin, the exact function of that protein is still not clear. This domain is now known to be in at least a hundred different proteins.
36p21ras to erk - a key signalling pathway GTP exhanges with GDPAssociationvia ras bindingdomainRas(GDP)Ras(GTP)SOSOUTINrafPPhosphorylationGRB2(adaptor with bothSH2 and SH3)SH2 binds to P-YGuanine nucleotideexchange factor;Bound to SH3 of GRB2Via poly-prolineMEKThe p21ras protein is a key signalling molecule in growth factor signalling. It acts similarly to other G proteins and is the original member of what are now known as small G proteins, or ras-like G proteins. It serves as a switch which is active when bound to GTP. Unlike heterotrimeric G proteins, the GTPase activity of p21ras is very low and it requires an associated protein, GAP, which is a GTPase activating protein. Active ras has a raf-binding effector site, which activates the serine/threonine kinase activity of raf. Raf phosphorylates MEK, which phosphorylates the mitogen- activated protein kinases (MAPK), erk1 and erk2. Thus MEK is a MAPK/erk kinase, but also known as a MAP kinase kinase, and raf is known as a MAP kinase kinase kinase.Phosphorylation(threonine/tyrosine)erk1/2PhosphorylationTranscriptionFactors
37A Genetic vs Molecular Description of the Ras Pathway From J. Downward’s article. Points out part of the vast complexity that is now known to regulate signalling pathways - where some sets of events were thought to proceed in a linear fashion, it is now known that there are multiple intersections at each point.
38PI 3-kinase phosphorylates PI(4,5)P2 in the plasma membrane Extracellular SignalTKR4,53,4,55P’ase3,4P85/p110PI3K?PDK2PDK1PKB308473NUCLEUSCYTOPLASMFrom a recent review article - Duronio, Biochem J.The Basics of PI 3-kinase Signalling
39Control of Cell survival by PI3K/PKB Attempts to highlight the many ways in which downstream targets that specifically affect cell survival can be altered by the activity of PI3K/PKB.Duronio,Biochem J, 2008
40Many Signalling Proteins May Act as Oncogenes When Mutated or OverexpressedPDGF ReceptorEGF Receptor (erb B)M-CSF Receptor (fms)PDGFEGF Growth FactorsM-CSFras proteinssrcTyrosine kinaseReceptorsGTP-bindingproteinsMembrane-associatedTyrosine kinasesNuclearproteinsMycfosjunThyroid HormoneReceptor (erb B)(raf)(fps/fes)Knowing mechanisms of signal transduction can explain many of the actions of oncogenes that are the causative agents of many types of cancers. Receptors that are mutated to be constitutively active can lead to continual growth of cells. Likewise, either tyrosine or ser/thr kinases that are normally responsive to growth factors can yield a growth signal when mutations cause them to be overactive. G proteins of the ras family are frequently mutated to cause them to remain in the GTP-bound or active state. Cells may overproduce growth factors that then act as a continual source of signalling. Finally, numerous transcription factors may be mutated to stimulate gene expression.CytoplasmicTyrosine kinasesCytoplasmicSer/Thr kinasesSteroid-typeGrowth factorReceptors
41REFERENCES – Signal transduction (Duronio, lecture 1) The Ins and Outs of Signalling. J. Downward. Nature 411: (2001).Kinome signaling through regulated protein-protein interactions in normal and cancer cells. T. Pawson and M. Kofler. Curr Opinion Cell Biol 21: (2009).Protein phosphorylation in signaling - 50 years and counting. T. Pawson and J. Scott. TIBS 30: (2005).Cell signaling by receptor tyrosine kinases. J. Schlessinger. Cell 103: (2000).Cell signaling by receptor tyrosine kinases. M.A. Lemmon and J. Schlessinger. Cell 141: (2010).V. Duronio, The Life of a Cell – Apoptosis Regulation by the PI3K/PKB Pathway. Biochem J. 415: (2008).For further in depth study: STKE, Science website
42Mechanisms of Signalling by RTK’s A. PKB Activation by PhosphorylationB. PI3K activation by pY binding and localization to plasma membraneC. PLCg activation by pY binding, phosphorylation and localization to membraneA. Binding of PH-containing enzymes PDK1 and PKB (also known as akt) to PIP3 at the plasma membrane result in a loss of autoinhibition, and together with phosphorylation of PKB by PDK1, and a second activating phosphorylation, changes PKB to a fully active kinase. B. In the case of type I PI 3-kinase, binding of SH2- containing p85 regulatory subunit alters conformation of the catalytic p110 subunit, so that it phosphorylates PI-4,5-P2 to yield PI-3,4,5-P3. C. In the case of PLC- gamma, binding of SH2 domains to phosphotyrosines of receptor leads to its tyrosine phosphorylation which is a key to its full activation.
43Multiple Effectors Regulated by RTK’s A. Sets of signalling pathways are simplified in this cartoon. Activation of some pathways can lead to feedback inhibition of earlier or parallel events. Many of the pathways impinge on transcription factors via numerous mechanisms, usually by direct phosphorylation, either to activate or inhibit activity. B. There are many ways of inhibiting or reversing a signal. EGF receptor can dimerize with a decoy receptor that does not allow signal transduction; phosphorylation of the receptor by other kinases can be inhibitory, or phosphatases can reverse phosphorylation events to inhibit signals. In the case of c-cbl, it acts to stimulate ubiquitination and degradation of targets it binds to (via its SH2 domain). Receptors can be internalized by endocytosis and eventually degraded (or recycled).