title: Rac2 regulation of phospholipase C-beta 2 activity and mode of membrane interactions in intact cells.
abstract: Phospholipase C-beta (PLCbeta) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCbeta(2) isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCbeta(2) with the plasma membrane. For either GFP-PLCbeta(2) or GFP-PLCbeta(2)Delta, a C-terminal deletion mutant lacking the region required for stimulation by Galpha(q), these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCbeta(2) and GFP-PLCbeta(2)Delta in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCbeta(2). Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCbeta(2), but had the opposite effect on GFP-PLCbeta(2)Delta, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCbeta isozyme by a GTP-binding protein and assign a novel function to the PLCbeta(2) C-terminal region, regulating its exchange between membrane-bound and cytosolic states.
authors.affiliation: Department of Pharmacology and Toxicology, University of Ulm, Germany.
authors.forename: Daria
authors.initials: D
authors.lastname: Illenberger
authors.forename: Claudia
authors.initials: C
authors.lastname: Walliser
authors.forename: Joachim
authors.initials: J
authors.lastname: Strobel
authors.forename: Orit
authors.initials: O
authors.lastname: Gutman
authors.forename: Hagit
authors.initials: H
authors.lastname: Niv
authors.forename: Verena
authors.initials: V
authors.lastname: Gaidzik
authors.forename: Yoel
authors.initials: Y
authors.lastname: Kloog
authors.forename: Peter
authors.initials: P
authors.lastname: Gierschik
authors.forename: Yoav I
authors.initials: YI
authors.lastname: Henis
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D054799:Phospholipase C beta; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D049452:Green Fluorescent Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; D054799:Phospholipase C beta; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein