26 Jun biopoblem set 4 – Following their activation upon binding to ligand,
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, theb2-adrenergic receptor (b2-AR), a GPCR, and its interaction withb arrestin are subjected to study. A cell line expressing theb2-AR is incubated in epinephrine, a ligand for this receptor, for 5 minutes. The cells are then lysed and theb2-AR is immunoprecipitated from the lysate. To determine ifb arrestin is bound to the receptor, the immunoprecipitate is examined by Western blotting using an antibody directed againstb arrestin. The experiment is repeated, but this time prior to and during epinephrine addition the cells are incubated in an inhibitor that blocksb2-AR phosphorylation by the kinase GRK2 (BARK). The data are shown below. (15 pts)
a. How does the activation state of the receptor affectb arrestin binding? What information do the kinase inhibitor studies provide aboutb arrestin binding to the receptor?
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b. Theb2-AR is prepared as the only integral membrane protein present in an artificial membrane preparation. Included in this preparation are either purified Gs protein or purified GRK2 or purifiedb 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 Gs activated? How does the presence of the kinase GRK2 affect Gs activation? How does the presence ofb arrestin affect Gs activation? How do the results with GRK2 relate to the findings in part (a) above? What can you deduce aboutb arrestin function from these data?
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c. When theb2-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) orb arrestin siRNA (see Chapter 8 of the text) to prevent expression ofb 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 theb arrestin–depleted cells compared to control cells?
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