Signal Transduction I

Question 1

What are the 4 classes of signals and what are their distinguishing features?

Answer 1

1. endocrine - released for widespread effects (release into blood)
2. paracrine - diffuses through extracellular medium and has effects on nearby cells
3. neuronal - delivered long distance but specifically to individual targets
4. contact - cells make direct contact via signaling molecules in their membranes

Question 2

What are the signal molecules released from paracrine glands called?

Answer 2

Local mediator

Question 3

What are first messengers?

Answer 3

natural extracellular ligands that bind and active receptors

Question 4

What are second messengers?

Answer 4

• Result from action of first messengers
• typically are small and generated by the signaling cascade

e.g. cAMP, cGMP, ions, lipids, NO

Question 5

What are the steps in signal transduction?

Answer 5

1. Primary transduction
2. relay
3. transduce and amplify (the second messenger)
4. integrate
5. Distribute
6. altered metabolism, shape or movement, gene expression of cell

Question 6

What is meant as a protein acting as a binary switch?

Answer 6

Proteins may be turned on or off depending on their environment

e.g. Ca2+ may bind to an enzyme and activate or deactivate it by changing its comformation

Question 7

How do proteins act as exchangers of information in signal transduction pathways?

Answer 7

One protein may detect the concentration of a substance and respond by activating another protein

e.g. cAMP dependent protein kinase detects cellular levels of cAMP and begins phosphorylating cellular targets

Note: the proteins don’t necessarily have to come into contact, the first protein may just detect the signal and send out a signal for the second protein in the cytoplasm

Question 8

How can a protein act as an information gate?

Answer 8

Multiple conditions must be met before the protein becomes active

Question 9

There are two types of cellular signaling pathways: those which alter protein function and those that alter protein synthesis. Of these two types which is faster and why?

Answer 9

• altering protein function through phosphorylation etc. is fast requiring from less than a second to minutes
• altering protein synthesis is slow because proteins must be transcribed and translated before they can be used

Question 10

What are the two broad types of receptors in signal transduction?

Answer 10

1. Cell surface receptors = transmembrane proteins that bind the signal on the outside of the cell
   e. g. Typrosine kinase, cytokine, CPCR
2. intracellular receptors - present in cytoplasm or nucleus
   e. g. receptors for the steriods

Question 11

T or F: agonists and antagonists can be chemically distinct or similar

Answer 11

T

Question 12

T or F: a cell can respond to signals even if they do not have receptors available that can bind the signaling ligand

Answer 12

False - cells must be able to bind the ligand in order to respond to its presence

Question 13

Why can cells only respond to a finite number of signals despite the presence of hundreds of signaling molecules in their environment?

Answer 13

Each cell only expresses certain receptors to respond to a few of the environmental cues

Question 14

Why are signal molecules ambiguous?

Answer 14

a single agonist ligand will have different effects depending on the tissue it binds.

e.g. Ach in heart causes decreased rate and force of contraction
Ach in salivary causes increases salivation
Ach in skeletal muscle induces contraction

Question 15

How can a single agonist have different effects in different cells?

Answer 15

• It may bind to a subtype of a given receptor that are coupled to different signaling pathways
• Expression of signaling proteins varies from cell type to cell type

Question 16

What determines the overall signaling profile in a given cell?

Answer 16

• combination of extracellular signals and expression of signaling proteins in a cell

Question 17

T or F: receptors exist in families of highly related proteins?

Answer 17

T

Question 18

How can one distinguish between receptor subtypes in a given receptor family?

Answer 18

• usually can be done using pharmacology (aka their ability to discriminate ligands that often have a very similar chemical structure)

Question 19

What are the three criteria for a physiological receptor?

Answer 19

1. Saturable - should be a finite number of receptors present
2. Specificity - binding of ligands to a receptor should parallel its ability to elicit a response via the receptor (if an agonist binds it should turn the receptor on and vice versa.
3. Kinetics - binding should be consistent with the time course of the biological effect by the ligand

Question 20

What are you looking for in a ligand binding assay and how is this performed for the reaction where:

[R] + [L] [R-L]

Answer 20

• you do equilibrium binding experiments to figure out what Kd (equilibrium dissociation constant) is

Kd = [L][R]/[R-L] = kback/kforward = molar units

Remember: Kd is the concentration at which half of the ligands have been bound (low Kd = high affinity)

• Typically radioactive ligands are used so binding can tracked

Question 21

Explain how a saturation binding assay experiment is completed?

Answer 21

1. varying amounts of radiolabeled ligand are added to different test tubes that all contain the same fixed number of cells
2. the test tubes containing cells are incubated until equilibrium is achieved
3. Each test tube is either (a) centrifuged (b) filtered
   4a. if centrifuged cells that have bound the radiolabeled ligand will sediment out and the amount of cells that have bound ligand in each tube can be measured
   4b. If centrifuged, cells that have bound the labeled ligand will not pass through the filter and the amount of these bound cells can be measured for each tube

Question 22

What is non-specific binding?

Answer 22

binding that does not take place at the receptor

e.g. binding to the test tube etc.

Note: the non-specific binding curve must be subtracted from the curve made from the raw data in the binding assay to give the specific binding curve which is the one that really matters

Question 23

What is the hallmark of ANY receptor?

Answer 23

Its ability to discriminate among closely related compounds.

Note: this is the basis of competition assays

Question 24

What is the purpose of competition binding assays?

Answer 24

• it defines a receptors pharmacology

- generates a rank order of potency (this is unique and acts as a receptor fingerprint)

Question 25

How are competition binding assays performed?

Answer 25

1. put in a fixed amount of an labeled antagonist ligand with a known receptor affinity
2. add increasing amounts of unlabeled test ligand
3. The antagonist ligand will be displaced if the receptor binds the test ligand
4. The amount of receptor bound to the labeled antagonist is measured and compared to the results that would be expected if not unlabeled test ligand were present

Question 26

What is EC50 in competition binding?

Answer 26

The effective concentration of competitor required to get 50% binding

Question 27

T or F: EC50 values are not comparable between experiments?

Answer 27

T

Question 28

Why are EC50 values not comparable between experiments?

Answer 28

because it depends on the amount of radioligand in the experiment

-in order to get some measure of comparison the EC50 must be converted to Kd values

Question 29

T or F: the rank order of potency (defined by a competition binding assay) unambiguously identifies a receptor subtype (aka acts as a fingerprint)

Answer 29

True

e.g if Iso>Epi>Norepi, then ß2 adrenergic receptor

If Iso>Epi = Norepi, then ß1 adrenergic

If Iso=Norepi>Epi, then ß3 adrenergic

Question 30

What is signal transduction?

Answer 30

• Takes a molecule from outside of the cell that binds to a protein receptor and turns it int an intracellular signal