Cellular Signaling Pathways

Home , Autocrine signaling, Paracrine signaling

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)

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 • 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 cAMP- response element (CRE). • CRE binds to the phosphorylated form of a transcription factor called CRE- binding protein (CREB) – A trans-regulatory element.

17 cAMP and CREB

• GPCRs and cAMP Recap! • Binding of hormones or neurotransmitters to Gs 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)

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 trans- regulatory 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 cis- regulatory 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.