Cellular Signaling I

Question 1

What is the general order of signaling events that occur in a cell

Answer 1

1. Ligand
2. GPCR
3. G protein effectors that are linked to second messenger generation
4. Regulator of G protein signaling (RGS)
5. Kinases
6. Arrsetins

Question 2

GPCR

Answer 2

G Protein Coupled Receptor

It resides in the plasma membrane with parts of the protein coming into the cell and sticking out of the cell.

They are the target of the ligands or many ligands interact with their part that sticks out of the membrane

Question 3

How does signaling takes place in GPCR

Answer 3

A ligand binds to the receptor, causes a conformational change to form a heterotrimer.

This conformation to heterotrimer promotes nucleotide exchange which activates the G protein receptor.

Normally in deactivated state the GPCR has GDP bound to it. But when activated the GDP is released and GTP is now bound to the receptor. This is what activates the protein.

Question 4

What happens when the GPCR is activated

Answer 4

The alpha intially is bound to the beta and gamma units but when GTP binds to the alpha unit it then dissociates from the beta and gamma subunits. It is now only bound to the GTP and acts downstream on its effector molecules

Question 5

What are the 2 downstream events

Answer 5

Cellular transcription or cellular response

Question 6

What is about the inactive bound state that the alpha, beta and gamma subunits do not interact with the effector molecules

Answer 6

Their active sites are shielded away as the active sites of the three subunits are not acting with each other (remember the fist and palm model)

Question 7

How are GPCRs specific

Answer 7

There are more than 800 GPCRs. They are very specific. They have different alpha, beta and subunits.

Question 8

Name the different alpha subunits

Answer 8

Galpha s - Activates adenylate cyclase
Galpha i 0 inhibits adenylate cyclase signalling

These two are antagonistic
Galpha q - activates the phospholipase C and finally Galpha 12/13 - activates small G proteins

Question 9

How can the same receptor produce different affects

Answer 9

Different ligands, different ligand concentrations can produce different results from the same receptor. This has to do with the Kd of the receptor with different ligands, it has different affinities for different ligands. This allows the cell to not respond in a binary fashion where the cell is simply on or off but in a differential fashion, in a graded manner

Question 10

How do you turn off G protein signalling

Answer 10

It is important to know that when alpha subunit is bound to GDP it is off and when it is bound to GTP subunit it is on.

The signal is turned off as the alpha subunit is also an eznyme and it cleaves the GTP unit into a GDP unit, releasing Pi and then it can reassociate with the beta and gamma subunits.

The activity is very slow.

There are also RGS proteins which bind to the alpha subunit and speed up the GTP hydrolysis.

Question 11

Basically what is a GPCR

Answer 11

It is a GEF, Guanine nucleotide Exchange Factor

Question 12

What is an RGS

Answer 12

GAPs, GTP Activating Proteins, any protein that speeds up the rate of hydrolysis of GTP is considered a GAP.

Question 13

What happens if the ligand is still bound to the receptor

Answer 13

The signalling cascade is initiated again

Question 14

What are the 2 types of desensitization events. What is the difference in the 2

Answer 14

Hetero and homologous desensitization of the receptors.

In homologous the mechanism only works on the receptors that generated the signal whereas heterologous will work on broad classes of receptors, even on different receptors.

In heterologous desensitization, the signalling doesnt have to come from GPCR but it can come from any signalling cascade that leads to the activation of PKA and PKC that phosphoryltes the GPCRs.

Question 15

What does a kinase do

Answer 15

Uses ATP to transfer a phosphate group on the substrate

Question 16

What happens in heterologous desensitization

Answer 16

PKA and PKC phosphorylates the GPCR which inhibits reassocaciation of the alpha, beta and gamma subunits. They are sterically hindered by the phosphorylation sites on the GPCRs.

Also the phosphorylated receptor now has a much lower affinity for the ligand.

Question 17

What happens in the homologous desensitization of the receptors

Answer 17

It is also a kinase mediated event, it is triggered by the recruitment of proteins called GRKs that phosphorylate that specific receptor.

When the surface of beta and gamma are exposed the interaction between the GRKs are favorable so the GRK binds to these 2 subunits and then it phosphorylates the receptor (at different sites than PKA and PKC).

Then arrestins bind to the phosphorylated receptors and sterically inhibits the reassocaition of alpha, beta and gamm subunits.

Arrestins also cause the internalization of the GPCR, it can either take the receptor off of the cell membrane or the ligand and the whole complex is degraded.
3rd thing can happens is that the receptor is remvoed, the G protein is dephosphorylated and then placed back into the membrane and now the alpha, beta and gamma subunits bind to the receptors in their inactive forms.

Question 18

Beta Andrenergic Receptors

Answer 18

Binds with the norepinephrine and epinephrine. These are the ones that trigger the fight or flight response.

Question 19

What does beta blocker do

Answer 19

It binds to the beta Andrenergic receptor. It physically the receptor, sounds like it is non competitive.

Question 20

What does cholera does to you

Answer 20

Leads to excessive diarrhea. This is done by the the cholera toxin. It ADP ribosylates the Galpha s receptor. In this from the receptor has no ligand bound to it but it is still activates. The alpha subunit however cannot hydrolyze the GTP molecule so this leads to a continuous signalling cascade resulting in excessive diarrhea.

Question 21

What happens in whooping cough

Answer 21

Remember that it is caused by Pertusis Bordetella. It releases the Pertusis toxin. This toxin leads to ADP ribosylation of the Galpha I (inhibitor) protein, so the alpha subunit cannot bind to the inhibitor molecule (or the receptor). This leads to downstream signal cascading events leading to whoping cough.

Question 22

Name one cogenital defects associated with G protein signalling

Answer 22

Jansen’s Metaphysical Chondrodysplasia in which the parathyroid receptors are activated with ligand binding to them, causes hypercalcemia due to bone resorption