Endocrine Abstracts

Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific hormones. Phosphatidylinositol 4, 5-bisphosphate (PtdIns(4, 5)P₂) has been shown to be critical for many endocytic processes including the internalization of G protein-coupled receptors (GPCRs). We have previously shown that depletion of PtdIns(4, 5) P₂ by the chemically induced plasma membrane recruitment of a 5-phosphatase domain prevents the internalization of the β₂ adrenergic receptor (β₂AR), as measured by its interaction with the early endosome marker Rab5. In this study we tested the effect of hormone-induced PtdIns(4, 5) P₂ depletion on β₂AR internalization with the help of type 1 angiotensin receptor (AT₁R).

We used luciferase-labelled β₂AR and fluorescently tagged Rab5 to follow the endocytic route of the receptors in HEK-293T cells using bioluminescence resonance energy transfer (BRET). To reduce plasma membrane PtdInsP₂ levels, we either applied our previously developed rapamycin-inducible heterodimerization system or we used wild type (wt) and internalization-incompetent mutant (Δ319 and TSTS/AAAA) forms of the G_q protein-coupled AT₁R which degrades PtdIns(4, 5) P₂ through the activation of phospholipase Cβ. We even created and tested an AT₁R fusion protein that is capable of both the rapamycin-induced and the hormone-activated depletion methods. We measured the rate of PtdInsP₂ depletion with the help of the PtdIns(4,5)P₂-binding PH domain of phospholipase Cδ₁.

Confirming our previous results, β₂AR internalization was inhibited after PtdIns(4, 5) P₂ depletion by our rapamycin-based system. A similar inhibition occurred after the activation of wt AT₁R. However, PtdIns(4, 5) P₂ depletion by internalization-incompetent AT₁R forms caused very little inhibition of β₂AR internalization, despite the higher rate of lipid depletion compared to the wt receptor.

Our data suggest that wt AT₁R might inhibit β₂AR internalization by competition for the endocytic machinery, and that the effect of plasma membrane PtdIns(4, 5) P₂ depletion on β₂AR internalization can be different depending on the method of lipid degradation, implying that distinct PtdIns(4, 5) P₂ pools might be required for endocytosis and receptor activation.