GPCRs 2

Question 1

What is the function of a GAP?

Answer 1

to promote GTP hydrolysis by providing an essential active site and promoting the correct positioning of the glutamine in switch domain II
can insert a + arginine into active site to help stabilise the transition state by interacting with - O atoms of phosphate

Question 2

Where do RGS proteins preferentially bind?

Answer 2

Ga-GTP to accelerate rate of hydrolysis and have amphipathic a-helices

Question 3

Where does RGS4 act and how?

Answer 3

activates GTPase of Gq specifically by certain receptors i.e. mAChRs

Question 4

How is RGS4 regulated?

Answer 4

PIP3 activated by the Gbg subunit interaction with PI-3Kinase

Question 5

what is the RGS4 cycle?

Answer 5

Gq->PLCb->IP3->Ca increase
RGS4 forms complex with Gaq-GTP and exerts GAP effects to shut off Ca
PIP3 then binds RGS4 and allows Ca to increase
Ca increase binds CaM and this competes with PIP3
Ca-CaM falls off when Ca is low and this allows reassociation with PIP3

Question 6

What are PDEs and what are some features?

Answer 6

breaks down cAMP although inhibition of PDE results in enhanced cAMP signalling effects
11isoenzyme
most drugs target PDE 5 which is activated by PKA and cGMP
some are activated by Ca (PDE1) some are PKA activated (PDE3,4,5)

Question 7

Upon endocytosis, what is the next steps for an internalised GPCR?

Answer 7

forms an endocytic vesicle which fuses with other vesicles to form an early endosome
early endosomes have low pH and this causes release of the receptor and ligand
the receptor can then be returned to the surface or degraded in a lysosome

Question 8

How does cholera toxin affect the GPCR?

Answer 8

disrupts GTPase activity of Gas and Gt and this causes Gs to be permanently activated increasing cAMP and PKA causing chloride efflux in the gut and rapid fluid loss.
this is done by the pathogenic CTA1 binding to ARF6 causing covalent modification by ADP-ribosylation to the arginine residue at the GTPase site

Question 9

How does pertussis toxin affect the GPCR?

Answer 9

the pathogenic S1 subunit is activate by factors like ATP and catalyses ADP-ribosylation at a cysteine residue of Gia making it incapable or exchanging GDP for GTP and blocking the cells inhibitory mechanisms
increases cAMP and insulin secretion and causes cough and reduced neutrophil and macrophage recruitment

Question 10

How can FRET be used to indentify cAMP concentrations?

Answer 10

a lot of flurophore transport is low cAMP where little transport is high cAMP