Signal Transduction I Flashcards
What are the 4 classes of signals and what are their distinguishing features?
- endocrine - released for widespread effects (release into blood)
- paracrine - diffuses through extracellular medium and has effects on nearby cells
- neuronal - delivered long distance but specifically to individual targets
- contact - cells make direct contact via signaling molecules in their membranes
What are the signal molecules released from paracrine glands called?
Local mediator
What are first messengers?
natural extracellular ligands that bind and active receptors
What are second messengers?
- Result from action of first messengers
- typically are small and generated by the signaling cascade
e.g. cAMP, cGMP, ions, lipids, NO
What are the steps in signal transduction?
- Primary transduction
- relay
- transduce and amplify (the second messenger)
- integrate
- Distribute
- altered metabolism, shape or movement, gene expression of cell
What is meant as a protein acting as a binary switch?
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
How do proteins act as exchangers of information in signal transduction pathways?
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
How can a protein act as an information gate?
Multiple conditions must be met before the protein becomes active
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?
- 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
What are the two broad types of receptors in signal transduction?
- Cell surface receptors = transmembrane proteins that bind the signal on the outside of the cell
e. g. Typrosine kinase, cytokine, CPCR - intracellular receptors - present in cytoplasm or nucleus
e. g. receptors for the steriods
T or F: agonists and antagonists can be chemically distinct or similar
T
T or F: a cell can respond to signals even if they do not have receptors available that can bind the signaling ligand
False - cells must be able to bind the ligand in order to respond to its presence
Why can cells only respond to a finite number of signals despite the presence of hundreds of signaling molecules in their environment?
Each cell only expresses certain receptors to respond to a few of the environmental cues
Why are signal molecules ambiguous?
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
How can a single agonist have different effects in different cells?
- 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
What determines the overall signaling profile in a given cell?
- combination of extracellular signals and expression of signaling proteins in a cell
T or F: receptors exist in families of highly related proteins?
T
How can one distinguish between receptor subtypes in a given receptor family?
- usually can be done using pharmacology (aka their ability to discriminate ligands that often have a very similar chemical structure)
What are the three criteria for a physiological receptor?
- Saturable - should be a finite number of receptors present
- 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.
- Kinetics - binding should be consistent with the time course of the biological effect by the ligand
What are you looking for in a ligand binding assay and how is this performed for the reaction where:
[R] + [L] [R-L]
- 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
Explain how a saturation binding assay experiment is completed?
- varying amounts of radiolabeled ligand are added to different test tubes that all contain the same fixed number of cells
- the test tubes containing cells are incubated until equilibrium is achieved
- 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
What is non-specific binding?
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
What is the hallmark of ANY receptor?
Its ability to discriminate among closely related compounds.
Note: this is the basis of competition assays
What is the purpose of competition binding assays?
- it defines a receptors pharmacology
- generates a rank order of potency (this is unique and acts as a receptor fingerprint)
How are competition binding assays performed?
- put in a fixed amount of an labeled antagonist ligand with a known receptor affinity
- add increasing amounts of unlabeled test ligand
- The antagonist ligand will be displaced if the receptor binds the test ligand
- 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
What is EC50 in competition binding?
The effective concentration of competitor required to get 50% binding
T or F: EC50 values are not comparable between experiments?
T
Why are EC50 values not comparable between experiments?
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
T or F: the rank order of potency (defined by a competition binding assay) unambiguously identifies a receptor subtype (aka acts as a fingerprint)
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
What is signal transduction?
- Takes a molecule from outside of the cell that binds to a protein receptor and turns it int an intracellular signal
