The languages of signal transduction- binding/dissociation

Question 1

What is a signalling network

Answer 1

1. Each cell is programmed to respond to specific combinations of intercellular signals
2. But only when it is appropriate
3. E.g. gene expression, secretion of hormone etc
4. Integration of multiple inputs allow different cellular responses

Question 2

Describe difference between fast and slow signal trasnduction

Answer 2

1. Fast
   - Energetically cheap as action is very fast
   - Transient process- can be removed in mins to hours
2. Or can be slow
   - Energetically costly
   - Stable- hours to years

Question 3

Where is the signal molecule present

Answer 3

1. Signal molecule either present in extracellular space or on surface of neighbouring cell

Question 4

Describe how a simple intracellular signaling pathway is activated by an extracellular signal molecule.

Answer 4

1. The signal molecule usually binds to a receptor protein that is embedded in the plasma membrane of the target cell.
2. The receptor activates one or more intracellular signaling pathways, involving a series of signaling proteins.
3. Finally, one or more of the intracellular signaling proteins alters the activity of effector proteins and thereby the behavior of the cell.

Question 5

How can effector proteins cause changes in cellular behaviour

Answer 5

1. Changes in shape of movement
2. Changes in gene expression
3. Altered metabolism

Question 6

What are the four forms of intercellular signalling

Answer 6

1. Contact-dependent signaling requires cells to be in direct membrane–membrane contact.
2. Paracrine signaling depends on local mediators that are released into the extracellular space and act on neighboring cells.
3. Synaptic signaling is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which are often located far away from the neuronal cell body.
4. Endocrine signaling depends on endocrine cells, which secrete hormones into the bloodstream for distribution throughout the body.

Question 7

What are characteristics of cell signalling

Answer 7

1. Enables transmission from outside of cell to nucleus or to cytoplasm
2. Fast ON and OFF (seconds to mins)
3. Energetically cheap (no protein synthesis)
4. Transient changes (minutes to hours) e.g shape, metabolism, mobility

Question 8

What are characteristics of gene expression

Answer 8

1. Enables transmission from outside of cell to nucleus
2. Slow ON and OFF (minutes to hours)
3. Energetically costly (transcription and translation)
4. Stable changes (hours to years)

Question 9

What are the different languages of signal transduction

Answer 9

1. Binding / dissociation- Interactions between proteins can dramatically affect many aspects of their behaviour, such as their activity and localization.
2. Conformational change - Proteins are not fixed in their three-dimensional shape or conformation; instead, they can often adopt multiple conformations that differ greatly in their activity.
3. Post translational modification – Proteins are subject to a number of different types of chemical modifications after they have been synthesized (these are collectively termed post-translational modifications).
4. Localisation within the cell - localization of proteins within the cell can affect their activities dramatically.

Question 10

Describe the activation of cyclic-AMP-dependent protein kinase (PKA).

Answer 10

1. not all intracellular signalling molecules are proteins! Some are small molecules called second messengers.
2. The binding of cAMP to the regulatory subunits of the PKA tetramer induces a conformational change,
3. causing these subunits to dissociate from the catalytic subunits
4. thereby activating the kinase activity of the catalytic subunits.
5. The release of the catalytic subunits requires the binding of more than two cAMP molecules to the regulatory subunits in the tetramer.
6. This requirement greatly sharpens the response of the kinase to changes in cAMP concentration.

Question 11

What are the main two types of PKAs in mammalian cells

Answer 11

1. type I is mainly in the cytosol
2. whereas type II is bound via its regulatory subunits and special anchoring proteins to the plasma membrane, nuclear membrane, mitochondrial outer membrane, and microtubules.
3. In both types, once the catalytic subunits are freed and active, they can migrate into the nucleus (where they can phosphorylate transcription regulatory proteins), while the regulatory subunits remain in the cytoplasm

Question 12

Describe signal transduction through binding/dissociation

Answer 12

1. A protein molecules physical interaction determines its biological properties
2. All proteins including signalling proteins bind to other molecules
3. Binding can be tight and long-lived or weak and short-lived
4. Always specific

Question 13

Describe the specificity of binding of proteins

Answer 13

1. The ability of a protein to bind selectively and with a high-enough affinity to a ligand to allow an interaction to occur depends on the formation of many weak noncovalent bonds.
2. Protein folding enables formation of a ligand binding site.
3. Complementary shapes of two interacting molecules allow many weak noncovalent bonds (hydrogen bonds, charged and hydrophobic regions, van der Waals attractions) to together form a highly specific interaction.
4. Reversible association between molecules (proteins, nucleic acids, lipids, inorganic compounds, peptides etc…) is central to signal transduction.

Question 14

What are the different types of protein interaction

Answer 14

1. Surface-string- Interacting protein has binding pocket in form of a cleft where interacting protein binds into like a string
2. Coiled-coil- Two helixes bind round each other, Very stable
3. Surface-surface- Very stable

Question 15

What does the modular nature of signalling proteins mean

Answer 15

1. Means composed of different domains
2. Domain- substructure produced by any contiguous part of a polypeptide that can fold independently of the rest of the protein
3. Domains are encoded by discrete regions of DNA
4. Typically 1 exon= 1 domain

Question 16

Give an example of the modular nature of signalling proteins

Answer 16

1. SRC-family kinases
2. 3 domains
3. SH3- Binds to string like stretches of polypeptide chain which contain proline residues
4. SH2- Binds to phosphor related tyrosine residues that are located in string like stretch
5. Kinase domain

Question 17

What are the consequences of protein-protein binding

Answer 17

1. Relocation of components
2. Increases efficiency and specificity
3. Alteration of activity

Question 18

How does binding reactions vary

Answer 18

1. Biologically important binding reactions can vary widely in their strength
2. ranging from weak and transient interactions that may be difficult
3. to detect to extremely strong interactions that are nearly as stable as covalent bonds.
4. Thus binding is not an all-or-none phenomenon, and a quantitative measure of the strength of binding interactions is needed in order to estimate whether they are likely to occur in the cell.

Question 19

What is used to measure the strength of association between two molecules

Answer 19

1. Affinity is a measure of the strength of association between two molecules.
2. The most commonly used measure of affinity is the dissociation constant (Kd) of the interaction—the ratio of unbound and bound species observed at equilibrium.
3. Affinity = the strength of association between two molecules. Defined by the equilibrium dissociation constant Kd.
4. Measures the relationship between molecule concentration and likelihood of interaction.

Question 20

What is Ligand binding assay

Answer 20

1. Method to detect and quantify the binding of a ligand to a molecular target
2. Biochemical assay, NOT a functional assay
3. Functional assay- allows us to measure consequence of binding
4. Molecular target has to be present on surface of cells- Intact cells Or isolated membrane fragments

Question 21

What is a Radioligand binding assay

Answer 21

1. Intact cells are immobilised to a filter support
2. Incubate filter membrane with radiolabelled ligand- contains radio isotope
3. Enables ligand to bind to receptor
4. Wash away unbound ligand
5. Transfer filter membrane into scintillation counter machine
6. This CONTAINS organic solvent and fluor molecule
7. Radioactivity – better radiation emitted is taken up by solvent and transferred to fluoroflor molecules
8. They get excited by energy from transfer of radiation and emit light

Question 22

What is happening at equilibrium

Answer 22

1. At equilibrium ligand binds and unbinds constantly

Question 23

How can you measure Kd

Answer 23

1. Need to measure the binding at equilibrium for different concentrations of the ligand
2. Get a saturation plot
3. X axis- ligand concentration
4. Y axis- fraction bound
5. Need to do control experiment to find nonspecific binding
6. Subtract nonspecific binding from total to find specific

Question 24

What are the parameters

Answer 24

1. Bmax= total concentration of specific binding sites for the ligand in a sample
2. Kd corresponds to the ligand concentration [L] when half of the proteins are occupied in equilibrium
3. Therefore, the smaller the equilibrium constant the more tightly bound the ligand is, or the higher the affinity between ligand and receptor

Question 25

What are the range of binding affinities

Answer 25

1. Low affinity - Weak interactions such as ubiquitin-ubiquitin receptor
2. Moderate affinity - Most protein interactions
3. High affinity - Antibody to antigen

Question 26

Describe affinity for endocrine system

Answer 26

1. Endocrine cells secrete hormones into bloodstream which are transported through bloodstream to target tissue
2. Bind to target
3. Hormone-receptor Kd:
4. Typically very low <10-9 M
5. Hormones are secreted into blood from a few cells - must work at low concentration.

Question 27

Describe affinity for synaptic interactions

Answer 27

1. Action potential reaches presynaptic membrane which induces releases of neurotransmitter molecules into synaptic space and bind to postsynaptic membrane to ion channels
2. Releases ion influx into target cell
3. Neurotransmitter-receptor Kd:
4. typically high >10-6 M
5. Neurotransmitters secreted into specialised synaptic cleft:
6. High koff assists in ‘resetting’ the synapse

Question 28

Do cellular response does not require activation of all receptors

Answer 28

1. No
2. Concentrations of extracellular stimulus needed to elicit cellular responses are typically lower than the Kd for association with receptor.
3. This allows great control of cellular response in sub-Kd ranges of ligand.
4. Physiological response occurs at low ligand concentration- lower than Kd