Purified M1 muscarinic cholinergic receptor and Gq/11 were coreconstituted in lipid vesicles. Addition of purified phospholipase C-beta 1 (PLC-beta 1) further stimulated the receptor-promoted steady-state GTPase activity of Gq/11 up to 20-fold. Stimulation depended upon receptor-mediated GTP-GDP exchange. Addition of PLC-beta 1 caused a rapid burst of hydrolysis of Gq/11-bound GTP that was at least 50-fold faster than in its absence. Thus, PLC-beta 1 stimulates hydrolysis of Gq/11-bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, Gq/11. GTPase-stimulating activity was specific both for PLC-beta 1 and Gq/11. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G proteins in vitro with fast physiologic deactivation of G protein-mediated signaling.

Three isoforms of phospholipase C, either PLC-beta 1, PLC-gamma 1, or PLC-delta 1, were added to the aqueous subphase beneath phospholipid monolayers formed at an air-solution interface, and the initial rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate was measured after addition of 10 microM free Ca2+. The monolayers were formed from mixtures of phosphatidylcholine (65% PC), phosphatidylserine (33% PS), and phosphatidylinositol 4,5-biphosphate (2% PIP2). Increasing the surface pressure of the monolayer, pi, from 15 to 25 mN/m decreases the rate of hydrolysis 16-, 13-, and 5-fold for PLC-beta 1, PLC-gamma 1, and PLC-delta 1, respectively. The simplest interpretation of these results is that a portion of each of the enzymes of area Ap must insert into the monolayer, doing work pi Ap, prior to hydrolysis of PIP2; binding studies with simple model compounds of known cross-sectional area are consistent with this interpretation. Removing the monovalent acidic lipid PS from the monolayer decreases the initial rates of hydrolysis of PIP2 about 3-fold for each PLC isoform, which suggests that negative electrostatic surface potentials increase the PLC activity.

Phospholipase C-␥ (PLC-␥) isozymes are thought to be activated by receptor-induced tyrosine phosphorylation. Proteins that activate PLC-␥1 have now been purified from bovine brain and identified as members of the tau family of microtubule-associated proteins. Activation of PLC-␥ by tau was enhanced in the presence of unsaturated fatty acids such as arachidonic acid, saturated fatty acids being ineffective. Maximal (15-20-fold) activation was apparent in the presence of 0.15 M tau and 25 M arachidonic acid (AA). The effect of tau and AA was specific to PLC-␥ isozymes in the presence of submicromolar concentrations of Ca 2؉ and was markedly inhibited by phosphatidylcholine. These results suggest that in cells that express tau, receptors coupled to cytosolic phospholipase A 2 may activate PLC-␥ isozymes indirectly in the absence of tyrosine phosphorylation through the hydrolysis of phosphatidylcholine to generate AA.The hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), 1 by a specific phospholipase C (PLC) is one of the earliest key events in the regulation of cellular function by more than 100 different extracellular signaling molecules (reviewed by Noh et al. (1995)). This reaction generates two intracellular messengers: inositol 1,4,5-trisphosphate (IP 3 ), which induces the release of Ca 2ϩ from internal stores, and diacylglycerol, which activates protein kinase C.Ten mammalian isoforms of PLC have been identified to date, and these can be divided into ␤ type (PLC-␤1, -␤2, -␤3, and -␤4), ␥ type (PLC-␥1 and -␥2), and ␦ type (PLC-␦1, -␦2, -␦3, and -␦4) enzymes on the basis of amino acid sequence (Noh et al., 1995). The distinct structural features of the different PLC types have been related to specific mechanisms of receptormediated enzyme activation. Thus, PLC-␥ isozymes are activated by tyrosine phosphorylation, and PLC-␤ isozymes are activated by heterotrimeric G proteins (Noh et al., 1995); the mechanism of PLC-␦ isozyme activation is not known.PLC hydrolyzes phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate as well as the physiological substrate, PIP 2 , in vitro, with PI-hydrolyzing activity often measured during PLC purification. While purifying PLC-␥1, the most abundant PLC isoform in brain cytosol, we observed that the PI-hydrolyzing activity of crude brain cytosol decreased more than expected on dilution. Furthermore, addition of crude cytosol to purified PLC-␥1 markedly enhanced PI-hydrolyzing activity. These observations suggested that brain cytosol contains a component that can enhance the activity of PLC-␥1 toward PI.We now describe the purification of this activator and its identification as the microtubule-associated protein tau. We also show that tau enhances the activity of PLC-␥1 toward PIP 2 to a markedly lesser extent than that apparent with PI and that the effect of tau on PLC-␥ activity toward PIP 2 is greatly increased in the presence of arachidonic acid (AA). Of the three types of PLC, the ␥ type isozymes are most sensitive to activ...