Following their activation upon binding to ligand, G protein coupled receptors,
ID: 178477 • 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 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 beta_2-adrenergic receptor (beta_2-AR), a GPCR, and its interaction with beta arrestin are subjected to study. a. A cell line expressing the beta_2-AR is incubated in epinephrine, a ligand for this receptor, for 5 minutes. The cells are then lysed and the beta_2-AR is immunoprecipitated from the lysate. To determine if beta arrestin is bound to the receptor, the immunoprecipitated is examined by Western blotting using an antibody directed against beta arrestin. The experiment is repeated, but this time prior to and during epinephrine addition the cells are incubated in an inhibitor that blocks beta_2-AR phosphorylation by the kinase GRK2 (BARK). The data arc shown below. How does the activation state of the receptor beta arrestin binding? What information do the kinase inhibitor studies provide about beta arrestin binding to the receptor? b. The beta_2-AR is prepared as the only integral membrane protein present in an artificial membrane preparation. Included in this preparation are either purified G_ protein or purified GRK2 or purified beta arrestin, as indicated by + symbols on the figure below. Epinephrine is added as indicated and the GTPase activity of the preparation is then measured. Why is GTPase activity measured in this assay? Under what conditions is G_s activated? How does the presence of the kinase GRK2 affect Gs activation? How does the presence of beta arrestin affect Gs activation? How do the results with GRK2 relate to the findings in part (a) above? What can you deduce about beta arrestin function from these data? c. When the beta_2-AR is activated a signaling cascade is initiated that leads to cAMP dependent phosphorylation of protein X. Cells were transfected with control silencing RNA (siRNA) or beta arrestin siRNA (see Chapter X of the text) to prevent expression of beta 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. What is a likely explanation for the differing pattern of protein X phosphorylation in the beta arrestin-depleted cells compared to control cells?Explanation / Answer
a)
In the absence of the inhibitor, the receptor phosphorylation allows the binding of beta-arrestin to the beta-adrenergic receptor. The current study diagram shows that beta-arrestin binding occurs when there is no inhibitor. If the inhibitor is absent, binding of ligand occurs and hence the presence of beta-arrestin bound to the receptor beta-adrenergic receptor happens when the inhibitor is absent. This binding of beta-arrestin will stop the overstimulation of the receptor by epinephrine.
b)
Beta-adrenergic receptor kinase is stimulated by protein kinase A and it targets the beta-adrenergic receptor. It is the method by which the cell will not respond to the epinephrine overstimulation due to its binding to beta-arrestin. Activation of beta adrenergic receptor by epinephrine activates Gs. G-alpha stimulates adenyl cyclase to synthesize cAMP which in turn activates cAMP-dependent kinase (Protein kinase A). The serine/threonine residues on beta adrenergic receptor kinase (beta-ARK) are phosphorylated by protein kinase A or cAMP dependent kinase. The beta-ARK will further phosphorylates serine/threonine residues on the beta-adrenergic receptors. The phosphorylation process of the beta-AR will facilitate the binding of beta-arrestin to the receptor. At this point, the epinephrine stimulation will not be able to activate Gs in the presence of beta-arrestin. Beta-arrestin is helpful in avoiding the overstimulation of beta-adrenergic receptor by epinephrine and aids in desensitization of cell.
The figure shows that presence of G-protein and epinephrine is enough to stimulate the GTPase activity.
c)
In the presence of beta-arrestin, the overstimulation of beta-AR by the epinephrine will not happen and gradually, the phosphorylation of proteinX will occur. But, if beta-arrestin is absent, the stimulation of beta-AR by the epinephrine continues and the receptor will not participate in the proteinX phosphorylation cascade. So, the phosphorylated protein should not be seen in the control with beta-arrestin. But, when the incubation with epinephrine increases, again the phosphorylation of proteinX stops. But, in the case of the absence or silencing of beta-arrestin, epinephrine presence could only reduce the intensity of phosphorylated proteinX.