In:
Journal of Cell Science, The Company of Biologists
Abstract:
Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate (PtdInsP2) has been shown to be critical for endocytosis of some cell surface receptors, such as EGF and transferrin receptor, but its role in G protein-coupled receptor internalization has not been investigated. By employing luciferase-labeled type 1 angiotensin II (AT1), type 2C serotonin (5HT2C) or β2 adrenergic (β2A) receptors and fluorescently tagged proteins (β-arrestin 2, plasma membrane targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK 293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdInsP2 in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdInsP2. Here we show that ligand-induced interaction of AT1, 5HT2C and β2A receptors with β-arrestin 2 was unaffected by PtdInsP2 depletion. However, arrival of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdInsP2. Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdInsP2 depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane but did not enter the cells. Our data suggest that in the absence of PtdInsP2, these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.
Type of Medium:
Online Resource
ISSN:
1477-9137
,
0021-9533
Language:
English
Publisher:
The Company of Biologists
Publication Date:
2012
detail.hit.zdb_id:
219171-4
detail.hit.zdb_id:
1483099-1
SSG:
12
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