Extracellular Regulated Kinase ERK Activation Cell Based Assay

Whereas GPCR-Ga fusion protein assays are per definition restricted to G protein-dependent signaling events, activation of the ERKs involves multiple signaling pathways including G protein-dependent and independent and P-arrestin-dependent pathways [45]. Most GPCRs are indeed able to activate the ERK pathway and thus, it may serve as a universal and broad-range readout of GPCR activation. A recently described cell-based assay that combines the homogeneous and nonradioactive properties of the alpha- screen technology to measure phosphorylation of ERK1 and ERK2, can be employed on endogenously expressed receptors and recombinant GPCRs and orphan 7TM proteins [57].

pH-Sensitive Probes: An Indicator of GPCR Internalization Agonist-promoted internalization is a regulatory phenomenon common to most GPCRs to limit receptor activation [58]. Activated GPCRs are rapidly phos-phorylated by GRKs followed by the recruitment of P - arrestins and the inter-nalization machinery. Several techniques have been developed to measure the disappearance of GPCRs from the cell surface and their accumulation in intracellular compartments. Recent strategies exploit the acidic nature of the endosomal compartments and the pH-sensitive cyanine dye CypHer 5, which is non-fluorescent at pH7.4 and fluoresces brightly in an acidic environment to measure receptor internalization [59, 60] . Surface - exposed receptors are labeled with CypHer 5-labeled antibodies, and ligand-dependent internaliza-tion into acidic endosomal compartments is monitored by the occurrence of intense intracellular fluorescence.

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