Stimulation of phospholipase C by guanine‐nucleotide‐binding protein βγ subunits

Abstract

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743 -7481. To investigate whether this stimulation was due to a soluble c1 subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein By dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that By subunits, purified from bovine retinal transducin (By,), markedly stimulated the hydrolysis of phosphatidylinositol4,Sbisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by by, was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin a subunit and was additive to activation of phospholipase C by guanosine 5'-[3-0-thio]triphosphate. byt also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to By,, but was also observed with highly purified subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in granulocytes. Only one of these forms was sensitive to stimulation by by,, demonstrating that stimulation of phospholipase C by by subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein By subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.The hydrolysis by phospholipase C of phosphatidylinositol 4,5-bisphosphate (PtdInsPJ to inositol 1,4,5-trisphosphate (InsP,) and diacylglycerol is a key mechanism by which many extracellular signalling molecules (hormones, growth factors and neurotransmitters) regulate the functions of their target cells [l, 21. There is ample evidence to suggest that many of the receptors interacting with these mediators stimulate phospholipase C via a guanine-nucleotide-binding protein (G protein) [3-51. In certain cell types, e.g.

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

“…The diffusion of ␤ 1 ␥ 2 -stimulated PLC␤ 2 (or PLC␤ 2 ⌬) is also much faster than that of PtdInsP 2 , which has D values of 0.5-1 m 2 /s (32, 34), enabling very fast spatial dispersal of the activated enzyme, not limited by the lateral diffusion of either lipidated protein targets or the substrate. This mechanism is likely relevant under physiological conditions, because PLC␤s are typically much less abundant (at least 100-fold) in cells than ␤␥ dimers (64,65), enabling sequential interaction of a single PLC␤ molecule with multiple ␤␥ dimers by dissociation and fast re-association. An important consequence of this fast diffusion is a dramatic increase in the travel range of ␤ 1 ␥ 2 -activated PLC␤ 2 (or PLC␤ 2 ⌬) to at least 1.9 m. This implies that stimulation by ␤ 1 ␥ 2 recruits PLC␤ 2 to act on dispersed PtdInsP 2 populations.…”

Nanomolar arachidonic acid influences the respiratory burst in eosinophils and neutrophils induced by GTP‐binding protein

“…The overall LDCL produced was only 5 -15 % lower than in the presence of 50 pM A13+, whereas the kinetics of the responses did not change (data not shown). On the other hand, it was reported that G,, subunits of pertussis-toxin-sensitive or insensitive Gproteins stimulated directly phospholipase C of bovine liver cytosol [39], of HL60 cytosol and membrane, of human and bovine peripheral neutrophils [40] or turkey erythrocytes [41]. Recently it has been shown that AlF,-is a potent activator of heterotrimeric G-proteins (G,,, GJ but has no action at all on low-molecular-mass G-proteins (p21 Ha-ra\, rap, rab and arf subfamilies) [42].…”

Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells

“…cyclase [4]~ facilitate the stimulation of type I1 and type IV adenylyl cyclase by activated ~x~ [4], regulate an unidentified effector moiety involved in the yeast pheromone response [8], and stimulate phospholipase C [9].…”