( esponse was quantitated using the Granu...)The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR

Agonist-induced internalization of CypHer5 labelled antibodies is concentration dependent. A sigmoidal dose response curve was obtained for each agonist by non-linear regression analysis. The dose response curves produced for EGFR and for each of the VSV-G-tagged β 2-adrenergic, δ -opioid, angiotensin II, and TRH receptors are shown in Figure 3. The VSV-G epitope tag provided improved results using CypHer5 when compared to those obtained with a c-myc epitope tag (data not shown, for further information contact incellanalyzer@amersham.com). The calculated EC₅₀ values were comparable to published data, where available (5).

Conclusion
In principle, CypHer5 pH-sensitive dye can be applied to the functional analysis and screening of any receptors that internalize into acidic endosomal vesicles upon ligand binding and activation. Dose response data were obtained for a range of different GPCR subtypes, producing acceptable EC₅₀ values. In addition it has been demonstrated that CypHer5 can be used to study the internalization of non-GPCRs. As an example, dose response data were generated for the tyrosine kinase receptor for EGF. Furthermore, for this assay, the use of a CypHer5 labelled antibody that binds an exofacial epitope of EGFR (rather than a tag) was shown. The data show that assays using CypHer5 pH-sensitive dye can be performed on the IN Cell Analyzer 3000 although pharmacologically relevant data can also be produced using the IN Cell Analyzer 1000 or a range of confocal microscopes.

References

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