Specificity and Structural Requirements of Phospholipase C-β Stimulation by Rho GTPases Versus G Protein βγ Dimers

Abstract

Phospholipase C-␤ 2 (PLC␤ 2 ) is activated both by heterotrimeric G protein ␣-and ␤␥-subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLC␤ isozymes with the rank order of PLC␤ 2 > PLC␤ 3 > PLC␤ 1 . The sensitivity of PLC␤ isozymes to Rho GTPases was clearly different from that observed for G protein ␤␥ dimers, which decreased in the following order: PLC␤ 3 > PLC␤ 2 > PLC␤ 1 for ␤ 1 ␥ 1/2 and PLC␤ 2 > PLC␤ 1 >>> PLC␤ 3 for ␤ 5 ␥ 2 . Rac1 and Rac2 were found to be more potent and efficacious activators of PLC␤ 2 than was Cdc42Hs. The stimulation of PLC␤ 2 by Rho GTPases and G protein ␤␥ dimers was additive, suggesting that PLC␤ 2 activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLC␤ 1 -PLC␤ 2 enzymes, ␤␥ dimers, and Rho GTPases are shown to require different regions of PLC␤ 2 to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLC␤ 2 were sufficient for efficient stimulation by ␤␥, the presence of the putative pleckstrin homology domain of PLC␤ 2 was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLC␤ regulators, which mediate PLC␤ isozyme-specific stimulation and are potentially involved in coordinating the activation of PLC␤ 2 by extracellular mediators in intact cells.Many extracellular signaling molecules elicit intracellular responses by activating inositol phospholipid-specific phospholipases C (PLCs), 1 which hydrolyze phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2 ) to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. These two second messengers modulate intracellular events through the regulation of intracellular free Ca 2ϩ and protein kinase C isozymes, respectively. The mammalian PLC isozymes can be divided into four major families: PLC␤, PLC␥, PLC␦, and PLC⑀ (1). The PLC␤ and PLC␥ subclasses have been shown to be regulated through G protein-coupled and protein-tyrosine kinase-linked receptors, respectively. The mechanisms by which PLC␦ isozymes and PLC⑀ are coupled to membrane receptors are less well understood (for recent reviews, see Refs. 1-4). Stimulation of PLC␤, of which four isozymes (PLC␤ 1 -PLC␤ 4 ) are known, is mediated by members of the ␣ q subfamily of G protein ␣ subunits and, excepting PLC␤ 4 , by G protein ␤␥ dimers (1-4). Activated ␣ q subunits stimulate PLC␤ in the rank order of efficacy of PLC␤ 1 Ն PLC␤ 3 Ͼ PLC␤ 2 . PLC␤ 4 is also activated by ␣ q subunits. The sensitivity of PLC␤ isozymes to ␤␥ dimers decreases in the order: PLC␤ 3

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

“…For example, this idea also corroborates studies by Illenberger and collaborators using chimera of PLC-β1/PLC-β2 which show that both the PH and the catalytic domain of PLC-β2 are required for Gβγ -activation [78].…”

“…Comparing a model structure of PH-β2 with the known structure of PH-δ1 suggests that the β5/β6 loop, which is longer than in PH-δ1, (Figure 3) could act as a regulatory domain for activation by Gβγ [41]. A peptide corresponding to this loop (PHβ [71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88] ) activates the enzyme, and this activation is competitive with Gβγ activation. The structure of PLC-β2 reveals that the β5/β6 loop does not belong to the surface that is tightly packed with the EF-hands and the catalytic regions, or to the hydrophobic ridge interacting with Rac2.…”

“…The ability of the peptide corresponding to PHβ [71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88] to activate the enzyme was unexpected.…”

Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ

“…Interestingly, recent reports have shown that PLCbs can also be activated by monomeric G proteins, such as Rho GTPases (Illenberger et al, 2003a;Snyder et al, 2003). Rho GTPases bind to the PH domains of PLCb and regulate the activity of each PLCb isozyme independently, and differently, from heterotrimeric G proteins (Illenberger et al, 2003a, b). Importantly, Rho GTPases are most active on the b2 isozyme of PLC, which is not expressed in mouse eggs (Dupont et al, 1996;Illenberger et al, 2003a).…”

“…Therefore, the possibility cannot be ruled out that fertilization might activate PLCb isozymes in a non-conventional manner, in which case this antibody would not block the sperm-induced [Ca 2+ ] i oscillations. Interestingly, recent reports have shown that PLCbs can also be activated by monomeric G proteins, such as Rho GTPases (Illenberger et al, 2003a;Snyder et al, 2003). Rho GTPases bind to the PH domains of PLCb and regulate the activity of each PLCb isozyme independently, and differently, from heterotrimeric G proteins (Illenberger et al, 2003a, b).…”