G–protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin–Darby canine kidney cells

Abstract

Membranes prepared from clone D1 of Madin–Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5′-(β-thiodiphosphate). While Ca2+ at 1 μM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2+-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4− incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.Key words: guanine nucleotide binding protein, phospholipase C, inositol phosphate, ras, Madin–Darby canine kidney cells.