Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation.
The pleckstrin homology domain of phospholipase Cdelta1 (PH(PLCdelta)) binds Ins(1,4,5)P(3) and PtdIns(4,5)P(2) specifically, and can be used to detect changes in Ins(1,4,5)P(3) in single cells. A fusion construct of PH(PLCdelta) and enhanced green fluorescent protein (EGFP-PH(PLCdelta)) associates with the plasma membrane due to its association with PtdIns(4,5)P(2). However, PH(PLCdelta) has greater affinity for Ins(1,4,5)P(3) than PtdIns(4,5)P(2), and translocates to the cytosol as Ins(1,4,5)P(3) levels rise. Prolonged activation of group I metabotropic glutamate receptor 1alpha expressed in Chinese-hamster ovary cells or endogenous M(3) muscarinic receptors in SH-SY5Y neuroblastoma cells gave an initial transient peak in translocation, followed by a sustained plateau phase. This closely followed changes in cell population Ins(1,4,5)P(3) mass, but not PtdIns(4,5)P(2) levels, which decreased monophasically, as determined by radioreceptor assay. Translocation thus provides a real-time method to follow increases in Ins(1,4,5)P(3). Graded changes in Ins(1,4,5)P(3) in Chinese-hamster ovary-lac-mGlu1alpha cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP-PH(PLCdelta) showed that changes in intracellular Ca(2+) concentration closely paralleled Ins(1,4,5)P(3) production. Moreover, Ins(1,4,5)P(3) accumulation and intracellular Ca(2+) mobilization within single cells is graded in nature and dependent on both agonist concentration and receptor density.
Nash
M S
MS
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, UK. msn2@le.ac.uk
Young
K W
KW
Willars
G B
GB
Challiss
R A
RA
Nahorski
S R
SR
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D016466:CHO Cells; D020013:Calcium Signaling; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate / Q000302:isolation & purification* / Q000378:metabolism*; D008164:Luminescent Proteins; D018613:Microscopy, Confocal / Q000379:methods; D010446:Peptide Fragments / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins / Q000378:metabolism; D043587:Receptor, Muscarinic M3; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001798:Blood Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; D043587:Receptor, Muscarinic M3; D018094:Receptors, Metabotropic Glutamate; D011976:Receptors, Muscarinic; D017981:Receptors, Neurotransmitter; C104077:metabotropic glutamate receptor type 1; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins