Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling
Abstract Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the interna...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2024-01-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-50732-x |
| _version_ | 1797363438043791360 |
|---|---|
| author | Dániel J. Tóth József T. Tóth Amir Damouni László Hunyady Péter Várnai |
| author_facet | Dániel J. Tóth József T. Tóth Amir Damouni László Hunyady Péter Várnai |
| author_sort | Dániel J. Tóth |
| collection | DOAJ |
| description | Abstract Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, β2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of β2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process. |
| first_indexed | 2024-03-08T16:21:22Z |
| format | Article |
| id | doaj.art-514428a6377948c388fa8fe3a7b24a47 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-08T16:21:22Z |
| publishDate | 2024-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-514428a6377948c388fa8fe3a7b24a472024-01-07T12:21:41ZengNature PortfolioScientific Reports2045-23222024-01-0114111410.1038/s41598-023-50732-xEffect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signalingDániel J. Tóth0József T. Tóth1Amir Damouni2László Hunyady3Péter Várnai4Department of Physiology, Faculty of Medicine, Semmelweis UniversityDepartment of Physiology, Faculty of Medicine, Semmelweis UniversityDepartment of Physiology, Faculty of Medicine, Semmelweis UniversityDepartment of Physiology, Faculty of Medicine, Semmelweis UniversityDepartment of Physiology, Faculty of Medicine, Semmelweis UniversityAbstract Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, β2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of β2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.https://doi.org/10.1038/s41598-023-50732-x |
| spellingShingle | Dániel J. Tóth József T. Tóth Amir Damouni László Hunyady Péter Várnai Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling Scientific Reports |
| title | Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling |
| title_full | Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling |
| title_fullStr | Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling |
| title_full_unstemmed | Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling |
| title_short | Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling |
| title_sort | effect of hormone induced plasma membrane phosphatidylinositol 4 5 bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling |
| url | https://doi.org/10.1038/s41598-023-50732-x |
| work_keys_str_mv | AT danieljtoth effectofhormoneinducedplasmamembranephosphatidylinositol45bisphosphatedepletiononreceptorendocytosissuggeststheimportanceoflocalregulationinphosphoinositidesignaling AT jozsefttoth effectofhormoneinducedplasmamembranephosphatidylinositol45bisphosphatedepletiononreceptorendocytosissuggeststheimportanceoflocalregulationinphosphoinositidesignaling AT amirdamouni effectofhormoneinducedplasmamembranephosphatidylinositol45bisphosphatedepletiononreceptorendocytosissuggeststheimportanceoflocalregulationinphosphoinositidesignaling AT laszlohunyady effectofhormoneinducedplasmamembranephosphatidylinositol45bisphosphatedepletiononreceptorendocytosissuggeststheimportanceoflocalregulationinphosphoinositidesignaling AT petervarnai effectofhormoneinducedplasmamembranephosphatidylinositol45bisphosphatedepletiononreceptorendocytosissuggeststheimportanceoflocalregulationinphosphoinositidesignaling |