title: D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation.
abstract: Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.
authors.affiliation: Cis Bio International, Research Department, B.P. 84175, F-30204 Bagnols-sur-Cèze Cedex, France. etrinquet@cisbiointernational.fr
authors.forename: Eric
authors.initials: E
authors.lastname: Trinquet
authors.forename: Michel
authors.initials: M
authors.lastname: Fink
authors.forename: Hervé
authors.initials: H
authors.lastname: Bazin
authors.forename: Florence
authors.initials: F
authors.lastname: Grillet
authors.forename: Fabrice
authors.initials: F
authors.lastname: Maurin
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Bourrier
authors.forename: Hervé
authors.initials: H
authors.lastname: Ansanay
authors.forename: Cédric
authors.initials: C
authors.lastname: Leroy
authors.forename: Audrey
authors.initials: A
authors.lastname: Michaud
authors.forename: Thierry
authors.initials: T
authors.lastname: Durroux
authors.forename: Damien
authors.initials: D
authors.lastname: Maurel
authors.forename: Fanny
authors.initials: F
authors.lastname: Malhaire
authors.forename: Cyril
authors.initials: C
authors.lastname: Goudet
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pin
authors.forename: Magali
authors.initials: M
authors.lastname: Naval
authors.forename: Olivier
authors.initials: O
authors.lastname: Hernout
authors.forename: Françoise
authors.initials: F
authors.lastname: Chrétien
authors.forename: Yves
authors.initials: Y
authors.lastname: Chapleur
authors.forename: Gérard
authors.initials: G
authors.lastname: Mathis
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry; D007295:Inositol Phosphates / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D007295:Inositol Phosphates; D043562:Receptors, G-Protein-Coupled; C002647:inositol 1-phosphate; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases