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 proteinsproteins 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 proteintyrosine 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 (G s ) 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 G s 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 G s 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 G s Ligand bound to the receptor α subunit is bound to GTP and dissociates from the the β and γ subunits. The G sα /GTP complex binds to and activates adenylyl cyclase. Adenylyl cyclase converts ATP to camp. GTP to hydrolyzed back to GDP. G sα binds to G βγ and inactivates adenylyl cyclase. β Adrenergic receptors and G s Amplification of the signal A single receptor-hormone complex causes the activation of at least 100 G s proteins. Each active G s 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 G s 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 G sα subunit is activated. β Adrenergic receptors and G i Some G proteins contain an α subunit which is inhibits adenylyl cyclase activity (G iα ). Some hormones interact with G s and some hormones interact with G i. 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 G sα 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 There are many many different G proteins associated with GPCRs. There are additional types of secondary messengers 16
camp and CREB We will now look at how camp regulates gene expression. cis-regulatory element A region of DNA or RNA that regulates the expression of genes located on the same molecule of DNA or RNA. trans-regulatory element Proteins that modify the expression of genes distant from the gene where it was transcribed. camp and CREB All genes regulated by camp contain a cis-regulatory element called campresponse element (CRE). CRE binds to the phosphorylated form of a transcription factor called CREbinding protein (CREB) A trans-regulatory element. 17
camp and CREB GPCRs and camp Recap! Binding of hormones or neurotransmitters to G s protein coupled receptors activates adenylyl cyclase. Adenylyl cyclase converts ATP to camp. camp and CREB Binding of camp to capk releases capk catalytic subunits. 18
camp and CREB Binding of camp to capk releases capk catalytic subunits. The catalytic subunits then translocate to the nucleus where it phosphorylates serine-133 on CREB camp and CREB Phosphorylated CREB binds to CRE and also interacts with a co-activator CBP/300 which permits CREB to stimulate transcription, 19
Receptor Tyrosine Kinases (RTKs) 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. Receptor Tyrosine Kinases (RTKs) RTKs consist of Ligand binding site Hydrophobic transmembrane α helix Cytosolic domain with tyrosine kinase activity. 20
Receptor Tyrosine Kinases (RTKs) Binding of a ligand causes RTKs to dimerize. The protein kinase of each receptor monomer then phosphorylates specific tyrosine residues of its dimer partner in a process called autophosphorylation The resulting phosphotyrosines serve as docking sites for other proteins in the signaling pathway Receptor Tyrosine Kinases (RTKs) Ras is a GTP-binding switch protein. Active when bound to a GTP Inactive when bound to a GDP Triggered by the binding of a hormone Activation is accelerate by the binding of a protein called guanine nucleotide exchange factor (GEF) Deactivation is accelerated by GTPase-activating protein (GAP) 21
Receptor Tyrosine Kinases (RTKs) Ras is a GTP-binding switch protein. Mutant Ras proteins bind to but cannot hydrolyze GTP. Results in Ras in a permanent on state and is associated with many types of cancer. Receptor Tyrosine Kinases (RTKs) The link between RTKs and Ras. Platelet derived growth factor (PDGF) and epidermal growth factor (EGF) are hormones which bind to RTK and activate Ras GRB2 acts as an adaptor protein An SH2 domain in GRB2 binds to a phosphotyrosine residue in the activated receptor. Two SH3 domains in GRB2 bind to and activate SOS. SOS functions as a gunanine nucleotide exchange factor (GEF) and helps convert inactive GDP- Ras to active GTP-Ras 22
MAP Kinase Pathways Protein kinase - transfers a phosphate group from ATP to a serine, threonine or tyrosine residue in a target protein. Mitogen - A chemical substance which promotes cell division. MAP kinase - Mitogen activated protein kinase MAP Kinase Pathways MAP kinases work in a series. MAP kinase kinase kinase (MKKK, MEKK, or MAP3K) is the first protein kinase in the series. MAP kinase kinase (MKK, MEK or MAP2K) is the second protein kinase in the series. MAP kinase is the third protein kinase in the series. 23
MAP Kinase Pathways Protein kinases are found downstream of activated Ras in the RTK signaling pathway. MAP Kinase Pathways Activated Ras binds to the N-terminal domain of Raf, a serine/threonine kinase. Raf binds to MEK, a protein kinase that phosphorylates both tyrosine and serine residues MEK phosphorylates and activates MAP kinase, a serine/threonine kinase. MAP kinase phosphorylates many different proteins including nuclear transcription factors. 24
MAP Kinase Pathways Experiment to determine the link between Raf and Ras. Constitutively active mutant Raf proteins induce quiescent cells to proliferate in the absence of hormone stimulation. A defective Raf protein cannot stimulate cells to proliferate by a mutant constitutively active Ras protein. These two experiments establishes a link between the Raf and Ras proteins. The next experiment proved conclusively that Ras and Raf interact with each other. MAP Kinase Pathways Experiment to determine the link between Raf and Ras. Yeast two-hybrid system. DNA binding domain (purple) fused to one of the interacting proteins referred to as the bait domain (pink) (in this case it is Ras) An activation domain of a transcription factor (orange) is fused to a protein expressed by a cdna library referred to as the fish domain (green) (in our case this is a cdna library which includes Raf) 25
MAP Kinase Pathways Experiment to determine the link between Raf and Ras. When the bait (Ras) and the fish (Raf) protein interact the DNA-binding domain binds to the UAS (upstream activating sequence) The activation domain stimulates assembly of the transcription-initiation complex (gray) and binds to the promoter (yellow) of the test gene (HIS) MAP Kinase Pathways Experiment to determine the link between Raf and Ras. The bait (Ras) plasmids have a Trp gene The fish (Raf) plasmids have a Leu gene Both plasmids are transfected into trp, leu and his mutant cells. Select for cells which grow in the absence of trp and leu. Plate selected cells on medium lacking His. Select for cells which grow on His deficient media Purify plasmids and determine which protein(s) bind to Ras. 26
MAP Kinase Pathways Experiment to determine at MAP kinase is downstream of Ras and Raf Mutate Ras and Raf in order to make them non-functional. Mutate MAP kinase so that it is constitutively active. Do not add a hormone Found that the cells proliferate. Conclusion is that MAP kinase is downstream of Ras and Raf MAP Kinase Pathways There are many different types of MAP kinase pathways in eukaryotic cells. Six MAP kinase pathways have been identified in Saccharomyces cerevisiae Triggered by extracellular signals 27
RTK and SRE We will now look at how RTKs regulate gene expression. Stimulation of the RTK-Ras signaling pathways causes the activation of MAP kinase. RTK and SRE Activated MAP kinase translocates to the nucleus and phosphorylates the C-terminal domain of transregulatory element called ternary complex factor (TCF) Phosphorylated TCF associates with two molecules of serum response factor (SRF) 28
RTK and SRE This trimeric DNA binding factor now binds to a cisregulatory element called serum-response element (SRE) activating transcription SRE regulates gene expression SRE is activated by the binding of many different growth factors to RTK. 29