Differential regulation of phospholipase C-beta2 activity and membrane interaction by Galphaq, Gbeta1gamma2, and Rac2.
We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-beta(2) (PLCbeta(2)) by G protein alpha(q) and betagamma dimers. We show that activation by alpha(q) and betagamma differ from activation by Rac2 and from each other. Stimulation by alpha(q) enhanced the plasma membrane association of PLCbeta(2), but not of PLCbeta(2)Delta, which lacks the alpha(q)-interacting region. Although alpha(q) resembled Rac2 in increasing the contribution of exchange to the FRAP of PLCbeta(2) and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLCbeta(2) by alpha(q) occurred by enhancing PLCbeta(2) association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by betagamma shifted the FRAP of PLCbeta(2) and PLCbeta(2)Delta to pure lateral diffusion 3- to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLCbeta(2) membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) in localized versus dispersed populations. PLCbeta(2) activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLCbeta(2) to act locally on PtdInsP(2) at specific domains. Activation by alpha(q) leads to lipid-like diffusion of PLCbeta(2) accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by betagamma recruits PLCbeta(2) to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP(2) populations.
Gutman
Orit
O
From the Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, and.
Walliser
Claudia
C
the Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Piechulek
Thomas
T
the Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Gierschik
Peter
P
the Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, Germany. Electronic address: peter.gierschik@uni-ulm.de.
Henis
Yoav I
YI
From the Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, and. Electronic address: henis@tauex.tau.ac.il.
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D036681:Fluorescence Recovery After Photobleaching; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054799:Phospholipase C beta / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D054799:Phospholipase C beta; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein