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PMID: 12771127 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Kinetic analysis of receptor-activated phosphoinositide turnover.

The Journal of cell biology ·Vol. 161 ·No. 4 ·2003-05-26 ·Pages 779-91

Xu C, Watras J, Loew LM

Abstract

We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCdelta1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

MeSH Terms
Bradykinin/pharmacology Cell Membrane/drug effects,metabolism Computer Simulation Green Fluorescent Proteins Kinetics Luminescent Proteins/metabolism Microscopy, Confocal Neuroblastoma/metabolism Phosphatidylinositols/chemistry,metabolism,pharmacology Receptors, Bradykinin/metabolism Tumor Cells, Cultured
Chemicals
Luminescent Proteins Phosphatidylinositols Receptors, Bradykinin Green Fluorescent Proteins Bradykinin
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Xu Chang
Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030, USA.
Watras James
Loew Leslie M
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.200301070
Published
2003-05-26
Pages
779-91
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2199375
Subset
IM
Grants
NCRR NIH HHS · P41 RR013186 · United States
NIGMS NIH HHS · GM35063 · United States
NINDS NIH HHS · NS40158 · United States
NCRR NIH HHS · RR13186 · United States
External Links
PubMed source DOI: 10.1083/jcb.200301070 PMC Full Text View journal details
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