Membrane binding of phospholipases C-beta 1 and C-beta 2 is independent of phosphatidylinositol 4,5-bisphosphate and the alpha and beta gamma subunits of G proteins.
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.
Runnels
L W
LW
Department of Physiology & Biophysics, State University of New York at Stony Brook 11794-8661, USA.
Jenco
J
J
Morris
A
A
Scarlata
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004735:Energy Transfer; D004910:Erythrocyte Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054799:Phospholipase C beta; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D008563:Membrane Lipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins