Chapter 15: Signal transduction Know the terminology: Enzyme-linked receptor, G-protein linked receptor, nuclear hormone receptor, G-protein, adaptor protein, scaffolding protein, SH2 domain, MAPK, Ras, protein kinase, MAPK, protein phosphatase, phospholipase, phosphodiesterase, camp, crosstalk,
Chapter 15: Signal transduction Outline: General principles of signal transduction Overview of: Signaling Receptors Transducers Targets Major types of cell-surface receptors RTK signaling G-protein signaling
General Principles of Signal Transduction 1. Communication usually involves a (i) signaling molecule, (ii) a receptor, (iii) intracellular signal transducers and (iv) targets
General Principles of Signal Transduction 2. Each cell responds to a complex profile of signaling molecules (crosstalk)
General Principles of Signal Transduction 3. Different cells respond differently to a particular signaling molecule
General Principles of Signal Transduction 4. Cells can remember the effects of some signals 5. Cells can adjust their sensitivity to a signal
General Principles of Signal Transduction 4. Cells can remember the effects of some signals 5. Cells can adjust their sensitivity to a signal
General Principles of Signal Transduction 6. Signal can exhibit complex responses to signal concentration
Signaling molecules Signaling molecules come in many chemical forms: Proteins: insulin, glucagon Steroids et al.: testosterone, estradiol, cortisol Amines: thyroxine, catecholamines, acetylcholine Gases: nitric oxide Signaling pathways require molecules with rapid rates of synthesis and degradation Typically released from one cell and recognized by another cell
Signaling molecules Secretory signals: Autocrine-signals affect same cell or cell type Paracrine-signals affect neighbouring cell Endocrine-signals affect distant cells Contact-dependent signals: -signals are not released but affect other cells in contact through protein-protein interactions
Autocrine signaling Signals released by one cell affect other cells in the immediate vicinity Amplify a response by inducing many like-cells to respond in the same way Allows cells to exhibit a coordinated response (a community effect)
Autocrine signaling
Paracrine signaling Signals released by one cell affect different cells in the immediate vicinity Synaptic transmission resembles paracrine stimulation but the response is limited to cells in very close proximity The outward propagation of the signal is limited by cellular uptake, extracellular degradation, and binding
Endocrine signaling Signals released by one cell affect different cells far away Endocrine signaling often exerts multiple effects on the organism by affecting many different tissues
Receptors Proteins that bind signals and initiate a signaling cascade Cell membrane receptors -integral membrane proteins that bind an extracellular signal and start a signal cascade Intracellular receptors -nuclear hormone receptors
Nuclear hormone receptors Examples that we have already discussed include steroid hormone receptor and thyroid hormone receptor NHRs are transcription factors that respond to specific ligands Ligands alter the ability to bind to specific DNA regulatory elements
Intracellular signal transduction Once the receptor is activated, the signal is propagated by proteins that act as: Relay proteins Messenger proteins Adaptor proteins Amplifier proteins Transducer proteins Bifurcation proteins Integrator proteins Latent gene regulatory proteins
Intracellular signal transduction Activated cell membrane receptors can alter the activity of intracellular enzymes including: Protein modifying enzymes kinases/ phosphatases acetylases/ deacetylases Lipid modifying enzymes Phospholipases Phosphotidyl inositol kinase Nucleotide modifying enzymes cyclases/ phosphodiesterases
Protein kinases
Phospholipases PLC generates DAG and phosphoinositides, such as IP3 (inositol 1, 4, 5- triphosphate)
Targets The final targets of signaling cascades are usually proteins: Regulators of gene expression (transcription factors, histone remodeling enzymes) Enzymes (metabolic enzymes) Structural proteins (cytoskeletal proteins) Effects alter activity (catalytic, DNA binding) or the ability to interact with other proteins (structural proteins, subcellular localization).
Cell surface receptors 3 main classes of cell surface receptors: Ion-channel linked receptors Enzyme linked receptors may possess intrinsic enzyme activity or, once ligands bind, activate enzyme activity G-protein linked receptors are trimeric GTPbinding protein (G-protein) that regulate the activity of other proteins
Enzyme-linked receptors 5 main classes distinguished by: type of effector (e.g. kinase vs. phosphatase) target (serine/threonine, tyrosine, histidine) type of linkage between receptor and enzyme 1. Receptor tyrosine kinase (-RTK) 2. Tyrosine kinase linked receptor 3. Receptor serine/threonine kinase 4. Receptor guanylyl cyclase 5. Histidine-kinase associated receptors
Receptor tyrosine kinases Most common type of receptor for many common protein hormones including EGF, PDGF, FGF, HGF, IGF-1, VEGF, NGF.
Receptor tyrosine kinases Receptor itself possesses intrinsic tyrosine kinase activity Once the ligand binds, the receptor can dimerize and it become an active tyrosine kinase It phosphorylates itself (autophosphorylation), causing: 1. Increase kinase activity 2. Increased affinity for other proteins Once bound, these docking proteins can become phosphorylated
Ligand-dependent autophosphorylation and docking
Ligand-dependent autophosphorylation and docking
Docking of intracellular proteins on phosphotyrosines Phosphotyrosine domains are binding sites for many different proteins with SH2 (=PTB) domains These can be enzymes (e.g., PLC, PI3K) or they can act as adaptor molecules to bind other proteins
Linking RTK to Ras and the MAPK cascade Once an adaptor protein (e.g., Grb2) binds to the RTK, it attracts another protein - Ras GEF (guanine nucleotide exchange factor) Ras GEF induces Ras to exchange its GDP for GTP (activating Ras). Active Ras then activates MAPKKK, which phosphorylates and activates MAPKK, which phosphorylates and activates MAPK, which phosphorylates many proteins, including transcription factors.
Activation of Ras
Activation of MAPK cascade
Scaffolding proteins help organize MAPKs
Summary: Enzyme-linked receptors How do enzyme-linked receptors generate variable cellular responses? Multiplicity of players (receptors, kinases etc) arise from gene duplication and divergence Recognize the critical role of phosphorylation/ dephosphorylation control as molecular switches Adaptor molecules allow construction of protein signaling cascades with variable outputs
G-protein linked receptors Ligand: Diverse ligands, such as epinephrine Receptor: Integral membrane protein with 7- transmembrane domains G-protein: Trimeric protein (α, β, γ) attached to the cell membrane by lipid anchors Effectors: Target proteins that show altered activity when they interact with activated G- protein subunits (α, or βγ)
G-protein linked receptors and G-proteins Receptor G-protein
Interaction between receptor and G-protein Once the ligand binds, the activated receptor recruits a G-protein Nucleotide exchange occurs (GTP replaces GDP) and the trimer dissociates into 2 parts: -α subunit -βγ subunit Both parts can regulate downstream pathways
G-protein dissociation
GTP hydrolysis ends signaling and induces trimerization
G s proteins are stimulatory Upon dissociation, a G s protein stimulates an effector enzyme, such as adenylate cyclase Adenylate cyclase converts ATP to camp Elevated camp stimulates camp-dependent protein kinase (PKA) by inducing the release of inhibitory subunits
PKA activation by camp
PKA activates gene expression
Inactivation of PKA pathway The G-protein -PKA pathway is inactivated by: Receptor desensitization GTP hydrolysis in G-protein camp hydrolysis by phosphodiesterase PKA inhibition Phosphatase action on PKA targets Activation of an antagonistic pathway (G i )
G-proteins and phospholipases Some G-proteins activate PLC (phospholipase C), triggering formation of inositol triphosphate (IP3) and diacylglycerol (DAG)
DAG, IP3, Ca 2+ and signal transduction DAG: substrate for production of eicosanoids, potent signaling molecules including arachadonic acid activates PKC IP3: induces release of Ca 2+ from ER stores via IP3- sensitive Ca-channels Ca 2+ : Elevated Ca 2+ can activates PKC and CamK.
Interactions between G-proteins and RTKs