An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis.
A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr(4+), Ga(3+), Al(3+), Fe(3+), Lu(3+), and Sc(3+), were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [3H]inositol phosphate production in cells preincubated with [3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.
Liu
Jay Jie
JJ
Lead Discovery Department, AstraZeneca Pharmaceuticals LP, Wilmington, DE 19850, USA. jay.lui@astrazeneca.com
Hartman
Deborah S
DS
Bostwick
James Robert
JR
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D051379:Mice; D010716:Phosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D018040:Receptors, Neurokinin-1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D015203:Reproducibility of Results; D012588:Scintillation Counting; D014316:Tritium / Q000032:analysis; D015040:Zirconium / Q000737:chemistry
D007295:Inositol Phosphates; D008670:Metals; D010716:Phosphatidylinositols; D018040:Receptors, Neurokinin-1; D014316:Tritium; D015040:Zirconium; D017479:Receptors, Platelet-Derived Growth Factor