Membrane Binding of Phospholipases C-β₁ and C-β₂ Is Independent of Phosphatidylinositol 4,5-Bisphosphate and the α and βγ Subunits of G Proteins

Abstract

We have measured the membrane binding affinities of purified phosphatidylinositol-specific phospholipases C-beta 1 and C-beta 2 to membranes of varying lipid composition using fluorescence methods. Our studies show that these proteins bind with affinities of 10(-5)-10(-4) M, with a small dependence on lipid type. Binding was relatively insensitive to the presence of phosphatidylinositol-specific phospholipases C-beta s' major physiological substrate, phosphatidylinositiol 4,5-bisphosphate, as well as the presence of Ca2+, which is required for activity. The presence of purified GTP gamma S-activated alpha 11 subunits of heterotrimeric guanine nucleotide binding proteins (G proteins) did not alter the membrane binding affinity of phosphatidylinositol-specific phospholipases C-beta 1, even though alpha 11 is a potent activator of this protein. Similarly, the presence of purified beta gamma subunits of G proteins did not alter the membrane association of phosphatidylinositol-specific phospholipases C-beta 2 even though these subunits strongly activate this isoform. These results argue against a recruitment model for PLC-beta activation by G proteins, negatively charged lipids, Ca2+, or substrate, and suggest that activation occurs through association of the membrane-bound species.

The Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Binds Strongly to Phosphatidylinositol 4,5-Bisphosphate

“…Recombinant human PLC-␦ 1 expressed in Escherichia coli, a generous gift from Dr. Mario J. Rebecchi, was purified as described elsewhere (22). Recombinant PLC-␤ 1 expressed in Sf9 cells, a generous gift from Dr. Suzanne Scarlata, was purified as described elsewhere (23).…”

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…However, in contrast to PLC-δ1, PLC-β2 as well as PLC-β1, bind to membranes with high affinity but little specificity, independent of PI(4,5)P 2 [37][38][39] . Their isolated PH domains are unable to bind to Ins(1,4,5) P 3 [33] but, together with the C-terminal region [38], allow PLC-β1 and PLC-β2 to interact non-specifically with membranes [40].…”

“…This binding was thought to activate the PLC-βs by recruiting them to the membrane surface [62]. In contrast, heterotrimeric G proteins activate PLC-βs without promoting its binding to membranes [37,39] and also without affecting the calcium dependence of its activity [10,37,38]. Instead, the strength of activation of PLC-β2 and -β3, at least by Gβγ subunits, appears to be directly proportional to the strength of association between the proteins [63].…”

“…However, it has been shown that PLC-β2 has high intrinsic binding affinity and is already bound to membranes before activation by Gβγ and thus activation does not occur by promoting PLC-β translocation [37][38][39]. More striking is the identification of soluble fragments of Gβγ, Gβ [86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105] and Gβ [42][43][44][45][46][47][48][49][50][51][52][53][54] , that are able to convey full activation of PLC-β2 [66,67,79].…”

WDR26 Functions as a Scaffolding Protein to Promote Gβγ-mediated Phospholipase C β2 (PLCβ2) Activation in Leukocytes

“…However, G␤␥ may directly stimulate the catalytic activity of these proteins. For example, it has been shown that translocation is not necessary for G␤␥-mediated PLC␤2 activation in vitro (14,15).…”