Signal Transduction Lecture Notes

Signal Transduction

General principles involve a stimulus (ligand) activating a receptor, which in turn activates an effector. Effectors can include G proteins or receptor kinases.

Overview of Signal Transduction

Signal transduction generally involves a receptor, a transducer, and an effector.

Extracellular Signals and Distance

Extracellular signals can act over short or long distances through several mechanisms:

  • Contact-Dependent Signaling: Requires direct contact between the signaling cell and the target cell. An example is Delta/Notch signaling, which leads to lateral inhibition and cell specialization. In this process, cells compete, and the cell with active Delta inhibits its neighbor from specializing likewise. Active Notch becomes inactive Notch.
  • Paracrine Signaling: Involves local mediators that affect target cells in the vicinity of the signaling cell. An example is branching morphogenesis of the lung, where FGF10, produced by mesenchyme cells, interacts with FGF10 receptors on bud epithelium cells. FGF10 production is inhibited by Sonic hedgehog (Shh), which is produced by epithelial cells at the tip of the growing bud. This leads to the creation of new centers of FGF10 production and the formation of new buds.
  • Synaptic Signaling: Occurs in the nervous system, where a neuron releases neurotransmitters across a synapse to target cells.
  • Endocrine Signaling: Involves hormones secreted by endocrine cells that travel through the bloodstream to reach target cells. An example is the response to dehydration, where osmoreceptors in the hypothalamus detect increased osmotic concentration in the blood. This leads to the release of antidiuretic hormone (ADH) from the posterior pituitary, which increases water retention by reducing urine volume and increases vasoconstriction to raise blood pressure. Includes the following steps:
    1. Sensory information goes to the neuroendocrine system which issues a command in the form of a chemical messenger (hormone).
    2. The hormone is transported to target cells via the bloodstream.
    3. The hormone reaches the target cells and binds to the cell receptors.
    4. The hormone-receptor complex triggers changes in the target cells.

Cellular Response to Extracellular Signals

Cells respond differently to different combinations of extracellular signals. Depending on the signals received, a cell can:

  • Survive
  • Grow and divide
  • Differentiate
  • Die (apoptosis)

The same extracellular signal can induce different responses in different cells. Various factors contribute to this:

  • Different receptors
  • Receptors with different combinations of subunits
  • Receptors coupled to different signaling pathways
  • Same signaling pathways with different outputs based on context

Adrenaline, for example, has diverse effects on different tissues due to:

  • Five different receptor subtypes (α1\alpha1, α2\alpha2, β1\beta1, β2\beta2, β3\beta3)
  • Several different mechanisms to transmit the signal inside the cell

Examples of adrenaline's effects include:

  • Liver: breakdown of glycogen to glucose
  • Adipose tissue: breakdown of triacylglycerol to fatty acids
  • Pancreas: inhibits insulin secretion, increases glucagon secretion
  • Muscle: inhibits insulin signaling and increases glycolysis
  • Heart: increases heart rate and contractility
  • Smooth muscle lining of blood vessels: muscle constriction
  • Lungs: bronchodilation, increased respiratory rate
  • Intestinal smooth muscles: inhibition of muscle fibers
  • Brain: vasodilation, arousal, fear, memory consolidation
  • Eyes: pupils dilate

Receptors and Signal Molecules

Most extracellular signal molecules bind to specific receptors. These receptors can be:

  • Cell-surface receptors: bind to hydrophilic signal molecules
  • Intracellular receptors: bind to small, hydrophobic signal molecules that can cross the plasma membrane

Intracellular Signaling Pathways

Multiple steps in intracellular signaling pathways are beneficial for:

  1. Signal modification (e.g., amplification, feedback)
  2. Interaction with other signaling pathways

These pathways can have:

  • Slower, long-term effects
  • Rapid, short-term effects

There are a limited number of receptors and signaling pathways:

  • A dozen receptor families
  • A few dozen intracellular signal transduction pathways

These pathways are conserved across species (yeast, worms, flies, fish, mice).

To terminate signaling, cells:

  • Deactivate/degrade receptors or signaling molecules
  • Use negative feedback mechanisms

Amplification

Different types of amplification can affect the magnitude and sensitivity of the response to a stimulus.

Identifying Genes in Signaling Pathways

Several methods can identify genes that act in signaling pathways:

  1. Genetic Screen
  2. Affinity Chromatography
  3. Genomics
  4. Cell reporter assay

1. Genetic Screens

Fruit Flies
  • Identified behavioral mutants of Drosophila using countercurrent distribution.
  • Identified photophobe (Ppb), a Drosophila mutant with a reversed sign of phototaxis.
  • Identified sevenless, a receptor tyrosine kinase (epidermal growth factor receptor).
  • Mutant at 24oC24^oC

Screens also used to identify suppressors and enhancers.

Nematodes

  • Screens in C. elegans identified Vulvaless (Vul) and Multivulva (Muv) mutants.

  • Identified lin-15 as a multi-vulva gene.

  • Identified let-60, where let-60(dn) is lethal and suppresses the lin-15 multi-vulva phenotype.
    Genetic screens in zebrafish identified genes involved in various developmental processes. In 1996, the Boston and Tubingen Screens:

  • Used 300 mutagenized males

  • Screened 5,000 F2 families

  • Identified 2,000 mutants

  • Involved 20 students and postdocs over 5 years

These screens identified many of the same genes as the fly and worm screens, as well as vertebrate-specific genes and pathways.

2. Affinity Chromatography

Affinity chromatography utilized to isolate receptors:

  1. Proteins from cell lysate.
  2. Proteins are added to Agarose beads with a Ligand on them (e.g. insulin).
  3. Proteins that don’t bind to the beads are eluted.
  4. The beads are washed
  5. Excess ligand is added to elute receptors from beads, or an enzyme is used is used to cleave the ligand/receptor complexes from the beads.
  6. SDS-PAGE is performed to isolate bound proteins for isolation and mass spectrometry to identify the proteins
SDS-PAGE for Protein Isolation
  1. Solubilize proteins from a cell lysate or protein solution using sodium dodecyl sulfate (SDS) and β\beta-mercaptoethanol
  2. Separate proteins based on size using polyacrylamide-gel electrophoresis (PAGE).

Coomassie dye stains proteins on a gel.

3. Genomics

Identify genes based on similarity to genes in the same species (paralogs) or different species (orthologs).

  • Olfactory receptors: Buck and Axel discovered these in 1991 and were awarded the Nobel Prize in 2004.

4. Cell Reporter Assay

Cell reporter assays can:

  • Identify the ligand of a receptor
  • Identify the receptor for a ligand

These assays require you to have already either identified a receptor or a ligand

Identifying Ligands of Receptors
  • Express an "orphan receptor" in cell culture and test ligands for activation of a cellular response.
  • Example: Discovery of orexin as a regulator of sleep and feeding, where HPLC is used to fractionate rat brain extract and identify fractions that activate cells expressing the orphan receptor.
Identifying Receptors for Ligands
  • Use a collection of cell lines that each expresses a different receptor.
  • Treat each cell line with the known ligand and test which cell line becomes activated.

Steps During Cell Signaling

  1. Production/modification/storage of signaling molecule
  2. Release of signaling molecule
  3. Transport in extracellular space
  4. Binding to receptor (usually on cell surface)
  5. Change in cellular function (short-term and/or long-term)
  6. Termination of the response

Key principles:

  • Target cells have specific receptors for signaling molecules.
  • A signaling molecule may bind to many different receptors.
  • Receptor activation can induce different effects in different cells.

Extracellular Signals

Signaling molecules are usually:

  • Small and hydrophilic
  • Proteins, small molecules, or gases

They are secreted from a source cell or displayed on its surface.

Molecules that bind to different receptors have different structures.

Competitor Molecules

  • Agonists: mimic the action of the natural ligand (e.g., muscarine mimics acetylcholine).
  • Antagonists: block the natural ligand from binding to the receptor (e.g., atropine).

Receptors and Signal Relay

Receptors relay signals into the cell through:

  • Receptor dimerization (e.g., receptor tyrosine kinase)
  • Conformational change (e.g., G-protein coupled receptor)

Receptors are highly specific for their ligands.

Classes of Cell-Surface Receptors

  1. Ion-Channel-Coupled Receptors:

    • Also called “transmitter-gated ion channels” or “ionotropic receptors.”
    • Ligands are neurotransmitters (e.g., glutamate, GABA, glycine).
    • Important for rapid signaling between neurons and between neurons and muscle.
    • Ligand binding causes transient opening of a channel in the receptor that allows ions to pass in or out of the cell.

  2. G-Protein-Coupled Receptors (GPCRs)

  3. Enzyme-Coupled Receptors