7. Following their activation upon binding to ligand, G protein-coupled receptor

Question

7. 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 ce Arrestins are proteins that bind to GPCRs and are involved in this desensitization. In order to understand GPCR-arrestin interactions, the B-adrenergic receptor (B-AR, a GPCR, and its interaction with Barrestin are subjected to study. A cell line expressing the B2-AR is incubated in epinephrine, a ligand for this receptor, for 5 minutes. The cells are then lysed and the B-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 B AR phosphorylation by the kinase GRK2 (BARK) The data are shown below. inhibitor hibitor B arrestin min in epinephrine: 0 5 0 5 a) How does the activation state of the receptor affect Barrestin binding? b) What information do the kinase inhibitor studies provide about B amestin binding to the receptor?

Explanation / Answer

Ques-a:

Usually beta arrestin produces desensitization of the receptors upon activation. So that receptor activation is leadg to waning of the responsiveness to the cell due to desnsitization for ligand binding. Arrestin binds to phosphorylated GPCR (G-protein coupled receptor) and blocks the further signalling media further signalling mediated by G-proteins and causes internalization of receptors, followed by activation of other G-protein independent signalling pathways.

Que-b:

This phosphorylation is predominantly leading to activation of beta arrestin protein activation in the cytoplasm and these proteins enhance uncoupling of heteromeric G proteins with GPCRs. The kinase inhibitors studies provide information that the beta-arrestin is going to bind efficiently with receptor so that initiation of signaling cascade occurs due to lack of "GRKs" for phosphorylation

Que-c:

Part-c: The reason for measuring GTPase activity is to unravel the “rate of GTP hydrolysis” when b 2 –Adrenoceptor activates G s so that the initial signal cascades are going to be measured when activated state is going through different cycles of GTP hydrolysis in the presence of ligand binding

Part-d: the addition of GRK2 is going to inhibit Gs activation and reverted in the presence of beta-arrestin

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