Cellular Signaling Pathways Signaling Overview • Signaling steps – Synthesis and release of signaling molecules (ligands) by the signaling cell. – Transport of the signal to the target cell – Detection of the signal by a receptor protein – A change in cellular metabolism, function or development. – Removal of the signal which terminates the response 1 Signaling Overview • Endocrine - hormones act on target cells which are distant from their site of synthesis. (examples: testosterone, estrogen, thyroid hormone….) • Paracrine - Signaling molecules released by a cell only affect those cells in close proximity to it. (examples: neurotransmitters, growth factors) Signaling Overview • Autocrine signaling - cells respond to substances they themselves release. (examples: growth factors and T-lymphocytes) • Membrane bound proteins- proteins on one cell can directly signal an adjacent cell. (example: embryo development) 2 Signaling Overview • Different cell types may have different sets of receptors for the same ligand each of which induces a different response. • The same receptor may occur on different cell types and binding of the same ligand may invoke a different response from each cell type. • Different ligand-receptor complexes can induce the same cellular response in some cell types. Four Classes of Receptors • G-protein coupled receptors – Ligand binding activates a G-protein which in turn activates or inhibits an enzyme that generates a secondary messenger or modulates an ion channel. – Examples: receptors for epinephrine, serotonin and glucagon, light activated receptors, receptors for neurotransmitters. 3 Four Classes of Receptors • Ion channel receptors – Ligand binding changes the conformation of the receptor to allow the movement of ions across the membrane – Example: receptors for acetylcholine Four Classes of Receptors • Tyrosine kinase linked receptors – Protein kinases: An enzyme which phosphorylates specific serine, threonine or tyrosine residues in target proteins. – Ligand binding causes the formation of a dimeric receptor. – The receptor itself does not have any inherent enzymatic activity so it links to and activates a protein-tyrosine kinase. – The dimer then activates one or more cytosolic protein- tyrosine kinases. – Example: receptors for human growth factor 4 Four Classes of Receptors • Receptors with intrinsic enzymatic activity. – Enzymatic activity activated by the binding of a ligand. – Example: Receptor serine/threonine kinases or also known as Receptor tyrosine kinases. • Autophosphorylates itself and can also phosphorylate various substrate proteins. – Ligands include Nerve Growth Factor (NGF), Platelet Derived Growth Factor (PDGF), Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), and insulin. – Regulate cell proliferation and differentiation, cell survival and regulation of cellular metabolism. G-Protein Coupled Receptors (GPCRs) • G-protein coupled receptors – Ligand binding activates a G-protein which in turn activates or inhibits an enzyme that generates a secondary messenger or modulates an ion channel. – Examples: receptors for epinephrine, serotonin and glucagon, light activated receptors, receptors for neurotransmitters. 5 G-Protein Coupled Receptors (GPCRs) • All GPCRs contain seven membrane spanning regions with the N-terminal segment on the exoplasmic face and the C-terminal segment on the cytosolic face of the plasma membrane G-Protein Coupled Receptors (GPCRs) • The actions of epinephrine and norepinephrin will be used as an example • Also known as adrenaline and noradrenaline. • Secreted by the adrenal glands and some neurons in response to stress. • Functions as both a hormone and a neurotransmitter. • Binds to two types of GPCRs in response to stress such as fright or heavy exercise. – β Adrenergic receptors – α Adrenergic receptors 6 G-Protein Coupled Receptors (GPCRs) • β Adrenergic receptors – Associated with Stimulatory G proteins (Gs) – Binding of epinephrine causes a rise in cAMP. – Found on the surface of liver and adipose tissue. • Binding of epinephrine causes the release of glucose and fatty acids. – Found on the surface of heart muscle • Binding of epinephrine causes an increase in heart rate contraction. – Found on the surface of smooth muscle in the intestine • Binding of epinephrine causes the smooth muscle to relax. G-Protein Coupled Receptors (GPCRs) • α Adrenergic receptors – Found on smooth muscle cells lining the intestinal tract, kidneys and skin. – Binding of epinephrine to these receptors causes the arteries supplying blood to these tissues to constrict. 7 β Adrenergic receptors and Gs • G proteins contain three subunits α, β and γ. • G proteins act as a GTPase switch. – “On” with GTP bound to the α subunit. – “Off” with GDP bound to the α subunit. β Adrenergic receptors and Gs • No ligand bound to the receptor – α subunit is bound to GDP and complexed with the β and γ subunits. 8 β Adrenergic receptors and Gs • Ligand bound to the receptor – α subunit is bound to GTP and dissociates from the the β and γ subunits. – The Gsα/GTP complex binds to and activates adenylyl cyclase. – Adenylyl cyclase converts ATP to cAMP. – GTP to hydrolyzed back to GDP. – Gsα binds to Gβγ and inactivates adenylyl cyclase. β Adrenergic receptors and Gs • Amplification of the signal – A single receptor-hormone complex causes the activation of at least 100 Gs proteins. – Each active Gs protein activates a single adenylyl cyclase. – Each adenyly cyclase catalyzes the synthesis of numerous cAMP molecules. • Binding of a single hormone molecule can result in several hundred cAMP molecules. 9 β Adrenergic receptors and Gs • Termination of the signal – The signal must stop when the concentration of hormones in the body decreases. – The receptors decrease their affinity to the hormone once the Gsα subunit is activated. β Adrenergic receptors and Gi • Some G proteins contain an α subunit which is inhibits adenylyl cyclase activity (Giα). • Some hormones interact with Gs and some hormones interact with Gi. 10 β Adrenergic receptors and Cholera toxin • Caused by the bacteria Vibrio cholerae. • Infection results in massive diarrhea caused by water flow from the blood through the intestinal epithelial cells into the lumen of the intestine. • Death by dehydration is common. β Adrenergic receptors and Cholera toxin • The toxin causes Gsα to remain in the active state since GTP cannot be hydrolyzed back to GDP. • This causes adenylyl cyclase to remain in the active state which in turn increases the concentration of cAMP molecules. • This rise in cAMP causes certain membrane proteins to allow the flow of water from the blood into the intestinal lumen. 11 β Adrenergic Receptors and Clinical Applications • Cardiac muscle has β adrenergic receptors which increase heart rate when bound to catecholamines such as epinephrine. • Drugs such as practolol as used to slow heart rate in cardiac arrhythmia and angina. • Practolo acts as an antagonist by binding to the receptor but not activating it. – Called a beta blocker. • Other drugs such as terbutaline bind to β adrenergic receptors but act as agonists which induce a response by mimicking the hormone epinephrine. – Terbutaline is used to treat asthma by causing the air passages of the lungs to open up Secondary Messengers • Secondary messengers amplify cell signals by increasing or decreasing their concentration after a ligand binds to a receptor. • cAMP (cyclic AMP) is a secondary messenger. – Results from the activation of adenylyl cyclase which converts ATP to cAMP. • The effects of cAMP are mediated through the action of cAMP-dependent protein kinases(cAPKs) also known as protein kinases A (PKAs). – Modifies the activities of target enzymes by phosphorylating specific serine and threonine residues. – Depending on the enzyme, can increase or decrease an enzyme’s catalytic activity 12 Secondary Messengers • cAMP-dependent protein kinases – Tetramers consisting of two regulatory (R) subunits and two catalytic subunits (C) – Binding of cAMP to the R subunits causes dissociation of the two C subunits which then phosphorylate specific proteins. Secondary Messengers • cAPKs regulate glucose/glycogen levels in the liver and muscle cells. • Glucose is stored as glycogen in liver and muscle cells. • Glucose is released from glycogen in response to a rise in the level of epinephrine. • Increases in cAMP increases the conversion of glycogen to glucose by inhibiting glycogen synthesis and stimulating glycogen degradation. 13 Secondary Messengers • An increase in cAMP activates cAPK. • Active cAPK phosphorylates and activates glycogen phosphorylase kinase (GPK) which then phosphorylates and activates glycogen phosphorylase (GP). • Active glycogen phosphorylase breaks down glycogen into glucose. • Active cAPK also phosphorylates and inactivates glycogen synthase (GS), which inhibits glycogen synthesis. • cAPK also inhibits phosphoprotein phosphatase(PP). Secondary Messengers • An decrease in cAMP inactivates cAPK. • Phosphoprotein phosphatase (PP) is no longer inhibited. • PP removes phosphate residues from GPK and GP resulting in inhibition of glycogen degradation. • PP also removes the phosphate from inactive GS, activating this enzyme and stimulating glycogen synthesis. 14 Secondary Messengers • Other secondary messengers produce the same glycogen response. Secondary Messengers • Secondary messengers can amplify a cell signal. 15 Secondary Messengers • The effects of cAMP on a cell depends on the type of cell and the type of cAPK. Secondary Messengers