lec 5: Cell signalling II

Describe fight or flight response in breif

epinephrine/ adrenaline act thro a g-protein coupled receptopr, to activate relay molecules (cAMP, 2 protein kinases), final activated protein is the enzyme glycogen phosphorylase, uses inorganic phosphate to release glucose monomers from glycogen in the form of glucose 1-phophate, amplifying pathway

final protein activated is

glycogen phophorylase enzyme

function of the glycogen phosphorylase enzyme

uses inorganic phosphate to release glucose monomers from glycogen in the form of glucose 1-phosphate molecules

GR: The fight or flight pathway is said to amplify hormonal signals

One receptor protein can activate approximately 100 molecules of G protein, and each enzyme in the pathway, once activated, can act on many molecules of its substrate, the next molecule in the cascade.

ligand receptor interaction

A specific ligand(key), bind to specific receptor (lock), with a specific pose in the active site, allow for specific action (cell response).

Agonist

binds to the receptor and produces an effect within the cell.

Antagonist

bind to the same receptor, but does not produce a response, instead it blocks that receptor to a natural agonist

Target cell receptor location

1. cell membrane

2. Cytosolic or nuclear

cell membrane proteins

most common, for hydrophilic or lipophobic ligands that can’t enter the cells (proteins, aa, peptides, gfs)

cytosolic or nuclear

hydrophobic or lipophilic molecules or very small ligands that can enter the cells (steroid hormones, nitric oxide)

Membrane receptors

1. ligand gated ion channels (ionotropic receptors)

2. G protein coupled receptors (metabotropic)

3. kinase linked receptors

Ligand gated ion channels

membrane channel receptor

has a region acting as a gate, closed in case of no ligand, when ligand binds conformational changes happen.

when a signaling molecule bind to receptor

channel opens, allow for the flow of specific ions (sodium, calcium), which affect cell activity.

when the signaling molecule dissociate

channel closes, and ions no longer enter the cell

example of ligand gated ion channels

1. Neural release of acetyl choline neurotransmitters (ligand) acting on nicotinic receptors (ligand-gated ion channels). allows the opening of sodium channels and flow of sodium in cells for neural communications

2. same thing, but the influx of sodium, stimulates the release of calcium ions for muscle contraction.

in example 1

Ligand: acetyl choline neurotransmitters

Ligand gated ion channels: nicotinic receptos

allow: entrance of sodium

response: action potential for neuron communication

essential in the nervous system function (and involved in learning and memory)

in example 2:

Ligand: acetyl choline neurotransmitters

receptor: nicotinic receptors

allow: entrance of sodium and release of calcium

response: skeletal muscle contraction

Type of gated channels

1. Ligand: specific chemical to open

2. voltage: specific gradient of electrical charge to open

3. Mechanical: specific tension to open

Voltage-gated examples

voltage gated calcium channels & voltage gated sodium channels

where are mechanical gated channels found

Around smooth muscle cells and artries

G-protein-coupled recptors (GPCRs)

most diverse family of membrane , involved in many physiolocal processes

1. immune system: cells such as T & B cells, and macrophages express various GPCRs

2. Cardiovascular system: cardiac muscle and blood vessels express sevral

3. Sensory organs: In sensory perception

what happens in case of malfunctioning GPCRs

many human disease including: cancer, cardiovascular, neurodegenrative disease (target for 50% of medicinal drugs)

1. beta blockers: target beta-adrenergic receptors

2. Histamine H1, H2 receptor Antagonists (ranitidine, telfast): Histamine H1, H2

Structure of GPCRs

• 7 transmembrane alpha helices

• extracellular domains for ligand binding

• intracellular domains interact with G proteins

• G proteins are heterotrimeric proteins (alpha, beta,, gamma subunits)

How do GPCRs work:

depends of G protein

inactive state: GDP bound to alpha subunit

Agoinist/ ligand bound: activate G protein GTP is attached

activated G protein= dissociate to alpha and beta-gamma subunit

activate downstream effector proteins to stimulate a response.

t or f Binding of signaling molecules is irreversible.

False, reversible

Kinase linked receptors

receptor tyrosine kinase:

1. Epidermal growth factor receptor

2. Vascular Endothelial gfr

3. Insulin receptor

Epidermal Growth Factor Receptor (EGFR)

On many skill types ex skin

involved in cell growth, proliferation and differentiation, important for skin development and repair

Vascular Endothelial Growth Factor Receptor (VEGFR)

In vascular endothelial cells (cells lining vessels)

critical for aangiogenesis

Angiogensis

For the formation of new blood vessels, essential for tissue growth and repair

Insulin receptor

On the surface of liver, muscles and fat cells

regulate glucose homeostasis

Biggest class of kinase linked receptors

Receptor tyrosine kinase, around 60 in the human body

Importance of RTKs

role in cell proliferation, diffrentiation and survival

Mutation in RTKs lead to

Uncontrolled cell groeth, associated with cancer

When does breast cancer have poor prognosis?

Excessive levels of receptor tyrosine kinase HER2, which is countered by the herceptin drug

Herceptin is

antagonist, bind to HER2 on cells and prevent cell division

Receptor tyrosine kinases (RTK)

RTK is a protein kinase—an enzyme that catalyzes the transfer of phosphate groups from ATP to another protein.

activated via dimerization when a gf is attached

may lead to 10 or more diff transduction pathways and cellular responses

regulate and coordinate many aspects of cell growth ,proliferation, reproduction

difference between RTKs and GPCRs

lies in the ability of RTKs simgle ligand binding that triggers many pathways.

steps of action of RTKs

1. dimerization which activates the tyrosine by adding a phophate from ATP

2. activate bound relay molecules

3. trigger a transduction pathway

4. cellular response

Signal transduction pathways

1. Protein phosphorylation and dephosphorylation

2. Secondary messanger

1. Protein Phosphorylation and Dephosphorylation

relay molecules in transduction are protein kinases

add phosphate to target protein making a process called phosphorylation cascade

phosphate is removed by

protein phophatases ex PTEN, (dephosphorylation turn off signal transduction pathway)

Small Molecules and Ions as Second Messengers

1. Second messanger (cAMP)

2. Calcium ions and inositol triphosphate IP3

Secon messanger cAMP

produced by adenylyl cyclase when ligand binds to GPCRs

cAMP then activates a variety of downstream effector proteins, as in the adenylyl cyclase cascade (amplify signals)

Calcium ions, Inositol triphosphate (IP3)

Function as second messengers

Example: Regulation of glycogen metabolism by epinephrine in fight/flight response

GPCR stimulation by epinephrine (ligand) leads to G protein activation → adenylyl cyclase activation (amplifier enzyme) → cAMP, (second messenger), activates protein kinase A → activation of phosphorylase kinase → activation of glycogen phosphorylase, which catalyzes the breakdown of glycogen to glucose1phosphate (response).

Response

a signal transduction pathway leads to the regulation of one or more cellular activities. The response at the end of the pathway may occur in the nucleus of the cell or in the cytoplasm.

Why different cells when they are exposed to the same signaling molecule, they have different responses?

The reason for signal specificity is that each cell type has different receptors, relay molecules, and effector proteins

Signal Termination

The cell has to stop responding to a particular signal in order to be able to respond to other signals, which is why all changes in the signal transduction pathway are reversible.

termination in reception

ligand dissociated from receptor

termination in transduction

phosphorylated/ active kinase are dephosphorylated

termination in response

effector proteins are dephosphorylated or degraded (beta catenin)

Common signalling pathways

1. JAK/ STAT

2. RAS/ RAF/ MAPK/ ERK

3. PI3K/AKT

JAK- STAT

for signaling cascade in cell communication

transmit signals from cell surface to nucleus in response to extracellular signals including: cytokines and growth factors that activate RTKs

cell growth, differentiation, and immune responses

(dysregulated in cancer → abnormal cell growth and differentiation).

STAT transcription factors

when cytokine ligands bind to RTK receptors, STAT binds to phosphotyrosine on receptor, where they are phosphorylated by the receptor associated JAK tyrosine kinases.

phosphorylated STAT proteins then dimerize and translocate to the nucleus, where they activate the transcription of target genes involved in cell growth, differentiation

Detect jak stat

qPCR, western blot, immunohistochemistry, ELISA

assess the functional consequences of JAK-STAT signaling (e.g., cell proliferation assay)

Medicine relatet to JAK STAT

: Tofacitinib (Xeljanz): a JAK1 and JAK3 inhibitor , primarily used for autoimmune conditions, and has been studied in clinical trials for various cancers, including solid tumors.

Ruxolitinib (Jakafi): Ruxolitinib is a JAK1 and JAK2 inhibitor primarily used for the treatment of myelofibrosis, a myeloproliferative neoplasm.

RAS/ RAF/ MAPK/ ERK

signal transduction activated downstream of both receptor and nonreceptor tyrosine kinases.

Key elements are serine/ thereonine kinases called MAP kinases (mitogen-activated protein kinases) activated in response to growth factors

regulate: cell growth, proliferation, differentiation, survival, and metabolism.

Step by step RAS/ RAF/ MAPK/ERK

activation of the GTP binding protein Ras protein (from rat sarcoma virus). ▪ Ras activates the Raf protein kinase, which in turn activates MEK and ERK. ▪ Activated ERK can translocate to the nucleus and phosphorylate transcription factors, altering gene expression involved in growth, proliferation and survival.

Drugs related to RAS/RAF/MAPK

RAF: Dabrafenib

MEK: Trametininb