Regulation of the rate and extent of phospholipase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins.
The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a simple model, we translated this apparent affinity to a bulk or three-dimensional equilibrium constant (Kd) and obtained a value of 3.2 microM. We confirmed this Kd by separately measuring the on and off (kf and kr) rate constants. The kf was slower than a diffusion-limited value, suggesting that conformational changes occur when the two proteins interact. The off rate shows that the PLC-beta 2.G beta gamma complexes are long-lived ( approximately 123 s) and that activation of PLC-beta 2 by G beta gamma would be sustained without a deactivating factor. The addition of alpha i1(GDP) subunits failed to physically dissociate the complex as determined by fluorescence. However, enzyme activity studies performed under similar conditions show that the addition of G alpha i1(GDP) results in reversal of PLC-beta 2 activation by G beta gamma during the time of the assay (30 s). From these results, we propose that G alpha(GDP) subunits can bind to the PLC-beta 2.G beta gamma complex to allow for rapid deactivation without complex dissociation. In support of this model, we show by fluorescence that G alpha i1(GDP).G beta gamma.PLC-beta 2 can form.
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Department of Physiology & Biophysics, State University of New York at Stony Brook 11794-8661, USA.
Scarlata
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004735:Energy Transfer; D004789:Enzyme Activation / Q000187:drug effects; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000502:physiology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D054799:Phospholipase C beta; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go