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 ?2-adrenergic receptor (?2-AR), a GPCR, and its interaction with ? arrestin are subjected to study. a. A cell line expressing the ?2-AR is incubated in the presence of epinephrine, a ligand for this receptor, for 5 minutes. The cells are then lysed and the ?2-AR is immunoprecipitated from the lysate. To determine if ? arrestin is bound to the receptor, the immunoprecipitate is examined by Western blotting using an antibody directed against ? arrestin. The experiment is repeated, but this time prior to and during epinephrine addition the cells are incubated in an inhibitor that blocks ?2-AR phosphorylation by the kinase GRK2 (BARK). The data are shown below. How does immunoprecipitation works? How does the activation state of the receptor affect ? arrestin binding? What information do the kinase inhibitor studies provide about ? arrestin binding to the receptor? - inhibitor + inhibitor ? arrestin min in epinephrine: 0 5 0 5

          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 ?2-adrenergic receptor (?2-AR), a GPCR, and its interaction with ? arrestin are subjected to study.
a. A cell line expressing the ?2-AR is incubated in the presence of epinephrine, a ligand for this receptor, for 5 minutes. The cells are then lysed and the ?2-AR is immunoprecipitated from the lysate. To determine if ? arrestin is bound to the receptor, the immunoprecipitate is examined by Western blotting using an antibody directed against ? arrestin. The experiment is repeated, but this time prior to and during epinephrine addition the cells are incubated in an inhibitor that blocks ?2-AR phosphorylation by the kinase GRK2 (BARK). The data are shown below. How does immunoprecipitation works? How does the activation state of the receptor affect ? arrestin binding? What information do the kinase inhibitor studies provide about ? arrestin binding to the receptor?
- inhibitor + inhibitor
? arrestin
min in epinephrine: 0 5 0 5

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 ?2-adrenergic receptor (?2-AR), a GPCR, and its interaction with ? arrestin are subjected to study.
a. A cell line expressing the ?2-AR is incubated in the presence of epinephrine, a ligand for this receptor, for 5 minutes. The cells are then lysed and the ?2-AR is immunoprecipitated from the lysate. To determine if ? arrestin is bound to the receptor, the immunoprecipitate is examined by Western blotting using an antibody directed against ? arrestin. The experiment is repeated, but this time prior to and during epinephrine addition the cells are incubated in an inhibitor that blocks ?2-AR phosphorylation by the kinase GRK2 (BARK). The data are shown below. How does immunoprecipitation works? How does the activation state of the receptor affect ? arrestin binding? What information do the kinase inhibitor studies provide about ? arrestin binding to the receptor?
- inhibitor + inhibitor
? arrestin
min in epinephrine: 0 5 0 5

Added by Michael C.

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