Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Abstract

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-␤ 2 (PLC␤ 2 ) by G protein ␣ q and ␤␥ dimers. We show that activation by ␣ q and ␤␥ differ from activation by Rac2 and from each other. Stimulation by ␣ q enhanced the plasma membrane association of PLC␤ 2 , but not of PLC␤ 2 ⌬, which lacks the ␣ q -interacting region. Although ␣ q resembled Rac2 in increasing the contribution of exchange to the FRAP of PLC␤ 2 and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLC␤ 2 by ␣ q occurred by enhancing PLC␤ 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 ␤␥ shifted the FRAP of PLC␤ 2 and PLC␤ 2 ⌬ 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 PLC␤ 2 membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) in localized versus dispersed populations. PLC␤ 2 activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLC␤ 2 to act locally on PtdInsP 2 at specific domains. Activation by ␣ q leads to lipid-like diffusion of PLC␤ 2 accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by ␤␥ recruits PLC␤ 2 to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP 2 populations. Phospholipase C-␤ (PLC␤)4 isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) to produce inositol 1,4,5-trisphosphate and diacylglycerol (1-3). They are activated to different extents by heterotrimeric G protein ␣ q subunits (␣ q ) and ␤␥ dimers (␤␥) (1-3). PLC␤ 2 is also activated by the Rho GTPases Rac and Cdc42 (4 -7). Activation by Rac has also been demonstrated for PLC␥ 2 (8). PLC␤ 2 has long been known to be expressed in hematopoietic cells (2, 3) but is also encountered in a variety of other cell types and tissues, including smooth muscle cells (9) and several brain regions (10, 11). Moreover, PLC␤ 2 was shown to be essential for taste perception via certain G protein-coupled oral taste receptors (12).Activation of PLC␤ 2 by ␣ q and related ␣ subunits requires the C-terminal region of the enzyme; mutants with deletions in this region (e.g. PLC␤ 2 ⌬, that lacks the Phe 819 -Glu 1166 segment) are resistant to stimulation by ␣ q , but undergo activation by ␤␥ and Rac/Cdc42 (7, 13-15). Recent results show that ␤␥ and Rac/Cdc42 activate PLC␤ 2 by interacting, at least in part, with different regions of the effector enzyme. Thus, although the pleckstrin homology (PH) domain of PLC␤ 2 is dispensable for activation of the enzyme by...

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

“…These affinity measurements are supported by a recent crystal structure of full-length PLC-␤3 and G␣ q showing minimal interaction between G␣ q and the C-terminal domain (18). Nevertheless, G␣ q -dependent activation of PLC-␤ isozymes at membranes clearly is supported by the C-terminal domain (23)(24)(25). Whereas it is reasonable to posit that membranes facilitate interactions between the C-terminal domain and G␣ q to support phospholipase activity, the nature of these potential interactions remains poorly understood.…”

“…The C-terminal domain also enhances the propensity of PLC-␤ isozymes to associate with membranes. This property was suggested by early cell fractionation studies showing shifts from particulate to soluble fractions of PLC-␤ isozymes that lacked an intact C-terminal domain (24,25). More recent fluorescent microscopy studies highlight the intrinsic propensity of this region to associate with cellular membranes, which is consistent with the observation that the C-terminal domain is highly electrostatically polarized along its long axis (50).…”

“…Indeed, G␤␥ does not appear to engage directly either of the autoinhibitory portions of PLC-␤ isozymes (41)(42)(43)(44)(45). Finally, the Rac GTPases also directly bind and activate PLC-␤2 and -␤3 (21,24,46,47). Because the site of Rac1 binding is far removed from the phospholipase active site, we presume that the Rac GTPases also activate the PLC-␤ isozymes using allosterism mediated by the membrane surface (37,48).…”

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

“…Baculoviruses containing cDNA for the human Gα i2 protein subunit [17] were donated from Professor Alfred G. Gilman (University of Texas Medical Center, Dallas, TX, USA), and baculoviruses containing cDNAs for the human Gβ 1 and bovine Gγ 2 subunits [18] were provided by Prof. Dr. Peter Gierschik (Institute of Pharmacology, University of Ulm). Cells were cultured at 27°C in Insect Xpress media (Lonza Group, Basel, Switzerland) containing 0.1 mg/ml of gentamicin.…”

Superantigen-Induced Steroid Resistance Depends on Activation of Phospholipase Cβ2

“…PLCb is activated by members of the Gaq family of G proteins (12), such as Ga11 (13) and Gaq (14). It was suggested that Ga11 acts upstream of PLCb activation in Jurkat T cells (8).…”