The Effect of Phosphatidylinositol 4,5‐bisphosphate Depletion on the Internalization of G Protein‐coupled Receptors

Phosphatidylinositol 4,5‐bisphosphate (PtdIns P 2 ) has been shown to be critical for many endocytic processes including the internalization of G protein‐coupled receptors (GPCRs). Our aim in this study was to compare the effect of different plasma membrane PtdIns P 2 depletion methods on GPCR internalization. We used bioluminescence resonance energy transfer (BRET) to follow the internalization of the luciferase‐tagged β 2 adrenergic receptor (β 2 AR) in HEK 293 cells. To reduce PtdIns P 2 levels, we applied either the rapamycin‐inducible recruitment of a 5‐phosphatase domain to the plasma membrane, or a truncated form of type 1 angiotensin receptor (AT 1 R) which activates phospholipase Cβ. We determined the rate of PtdIns P 2 degradation using the PH domain of phospholipase Cδ 1 which binds PtdIns P 2 specifically, and found it to be comparable for the two depletion methods. While PtdIns P 2 depletion by our rapamycin‐based system inhibited the internalization of β 2 AR, PtdIns P 2 depletion by AT 1 R had no effect on it, measured by the same method. Our data suggest that the effect of plasma membrane PtdIns P 2 depletion on the internalization of β 2 AR can be different depending on the method by which the lipid is degraded. Further investigation is needed to determine whether this discrepancy is due to degradation of distinct PtdIns P 2 pools of the plasma membrane or other factors are responsible for it. Support: OTKA K105006