title: Phospholipase C beta2 association with phospholipid interfaces assessed by fluorescence resonance energy transfer. G protein betagamma subunit-mediated translocation is not required for enzyme activation.
abstract: Phospholipase C beta2 (PLC beta2) is activated by G protein betagamma subunits and calcium. The enzyme is soluble and its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), is present in phospholipid membranes. A potential mechanism for regulation of this enzyme is through influencing the equilibrium association of the enzyme with membrane surfaces. In this paper we describe a fluorescence resonance energy transfer (FRET) method for measuring the association of PLC beta2 with phospholipid bilayers. The method allows equilibrium measurements to be made under a variety of conditions, including those that support enzymatic activity and ability to be regulated by G proteins. Using this method it was found that PLC beta2 bound to vesicles containing anionic lipids and demonstrated a selective and unique interaction with PIP2-containing vesicles. The FRET data were corroborated with a centrifugation based method for estimating the affinity of PLC beta2 for vesicles. Apparently different modes of association of PLC beta2 with vesicles of different composition can be distinguished based on alterations in resonance energy transfer efficiency. Association of PLC beta2 with PIP2 vesicles requires an intact lipid bilayer, is blocked by neomycin, and is not affected by D-myo-inositol 1,4,5-trisphosphate (D-IP3). G protein betagamma subunits do not alter the affinity of PLC beta2 for lipid bilayers and at the PIP2 concentrations used to measure betagamma-dependent stimulation of PLC activity, the majority of the PLC beta2 is already associated with the vesicle surface. Furthermore, under conditions where betagamma subunits strongly activate PLC activity, the extent of association with vesicles is unaffected by betagamma subunits or calcium. These results indicate that activation of PLC beta2 by G protein betagamma subunits or Ca2+ in vitro does not involve translocation to the vesicle surface.
authors.affiliation: Department of Pharmacology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: V
authors.initials: V
authors.lastname: Romoser
authors.forename: R
authors.initials: R
authors.lastname: Ball
authors.forename: A V
authors.initials: AV
authors.lastname: Smrcka
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004735:Energy Transfer; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054799:Phospholipase C beta; D010743:Phospholipids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018411:Spodoptera; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003902:Detergents; D007527:Isoenzymes; D008051:Lipid Bilayers; D019269:Phosphatidylinositol 4,5-Diphosphate; D010743:Phospholipids; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins