Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits.
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, 743-748]. To investigate whether this stimulation was due to a soluble alpha 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 beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t 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 beta gamma t, but was also observed with highly purified beta gamma 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 HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.
Camps
M
M
Pharmakologisches Institut, Universität Heidelberg, Federal Republic of Germany.
Hou
C
C
Sidiropoulos
D
D
Stock
J B
JB
Jakobs
K H
KH
Gierschik
P
P
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001923:Brain Chemistry; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006098:Granulocytes / Q000201:enzymology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D046911:Macromolecular Substances; D009504:Neutrophils / Q000201:enzymology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D007295:Inositol Phosphates; D007527:Isoenzymes; D046911:Macromolecular Substances; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins