Following their activation upon binding to ligand, G protein-coupled receptors,
ID: 256715 • Letter: F
Question
Following their activation upon binding to ligand, G protein-coupled receptors, GPCRs, in turn activate G proteins and thereby initiate a signaling cascade. Following ligand binding, GPCRS 4. become phosphorylated by G protein receptor kinases (GRKs) which, in turn, leads to desensitization of the receptor such that continued stimulation by ligand results in a waning responsiveness of the cell. Arrestins are proteins that bind to GPCRs and are involved in this desensitization. In order to understand GPCR-arrestin interactions, the B2-adrenergic receptor (B2-AR), a GPCR, and its interaction with B-arrestia are subjected to study.| a. A cell line expressing the ?2-AR is incubated in epinephrine, a ligand for this receptor, for 5 minutes. The cells are then lysed and the B2-AR is immunoprecipitated from the lysate. To determine if B arrestin is bound to the receptor, the immunoprecipitate is examined by Western blotting using an antibody directed against B arrestin, The experiment is repeated, but this time prior to and during epinephrine addition the cells are incubated in an inhibitor that blocks B2-AR phosphorylation by the kinase GRK2 (BARK). The data are shown below inhibitor inhibitor B arrestin min in epinephrine: 0 5 0 5 Make 2 conclusions b. when the ?2-AR is activated, a signaling cascade is initiated that leads to CAMR dependent phosphorylation of protein X. Cells were transfected with control silencing RNA (siRNA) or arrestin siRNA to prevent expression of ? arrestin. The cells were then incubated in epinephrine and, at various times, aliquots of the preparation were prepared for Western blotting using an antibody directed against phosphorylated protein X. The data are shown below Min in epinephrine: 02 5 1 20 30 control siRNA phosphorylated protein X B arrestin siRNA (i)List your observations. (ii)Make conclusions.Explanation / Answer
a) Two conclusion can be drawn :
1) In the absence of inhibitor when ligand epinephrin get bind to the B2-AR (GPCR) , beta arrestin also bind to receptor and GRK phophorylate the GPCR and uncouple the binding of ligand and also deactivates the receptor so that after 5 min. incubation of ligand (epinephrin) binding , a protein band of beta areestin was appear.
2) But in the presence of inhibitor, no phophorylation of GPCR (B2-AR) takes place by GRK and beta arrestin do not able to bind the receptor thus not able to desensitized the receptor which was shown by the absence of beta arrestin band in western blotting.
(i) Observation:
1) in control silencing initial cascade was intiated but after few minutes siRNA degrade m-RNA expression and further cascade was hampered and we get few downstream phophorylated proteins.
2) While in Beta arrestin siRNA complete degradation of beta-arrestin m-RNA was takes palce so that all the proteins get phosphorylated from the signal cascade.
Conclusion:
Beta -arrestin desensitized the receptor so that the cascading signalling process but when this protein was not expressed by silencing m-RNA expression then GPCR receptor remains sensitive to ligand and process the signalling and we get all phophorylated proteins.