Fig. 4.

PIP₂ does not increase Gαq transactivation by tubulin. A, The effects of carbachol and PIP₂ on the membrane association of tubulin are additive. Recruitment of tubulin to the membrane was studied using increasing concentrations of FM-tubulin. Membranes from Sf9 cells expressing m1 muscarinic receptors, Gαq, and PLCβ₁ were incubated with carbachol and FM-tubulin (at the indicated concentrations), with or without PIP₂. SDS-PAGE (50 μg of membrane protein in each lane) was followed by measurement of the fluorescence of membrane-associated tubulin with a fluorescence imaging system (Storm 840; Molecular Dynamics). The results represent one of three similar experiments performed in triplicate. Open circles, Membranes treated with 1 mm carbachol; filled circles, membranes treated with 1 mm carbachol and 30 μmPIP₂. B, PIP₂-assisted recruitment of tubulin–[³²P]AAGTP to the membrane is concentration-dependent. Membranes from Sf9 cells containing m1 muscarinic receptors, Gαq, and PLCβ₁ were incubated with 1 mm carbachol, 1 μmtubulin–[³²P]AAGTP, and increasing concentrations of PIP₂, as described in Figure 1. After SDS-PAGE (50 μg of membrane protein in each lane)³²P-labeled protein bands were measured by phosphorimage analysis. One of three identical experiments done in triplicate with similar results is shown. C, Carbachol-evoked Gαq transactivation by tubulin is not affected by PIP₂. The experiments were done as described in A, except that tubulin–[³²P]AAGTP was used. Proteins were resolved by SDS-PAGE (50 μg of membrane protein in each lane), and the radioactivity of the Gαq bands ([³²P]AAGTP was transferred from tubulin) was measured by phosphorimage analysis (Storm 840; Molecular Dynamics). One of five independent experiments done in triplicate with similar results is shown. Open circles, Membranes treated with 1 mm carbachol;filled circles, membranes treated with 1 mmcarbachol and 30 μm PIP₂. D, The increased membrane association caused by PIP₂ was not linked to Gαq transactivation. The percent ratios of [³²P]AAGTP-labeled Gαq and tubulin–[³²P]AAGTP at the various PIP₂ concentrations are derived from the experiment described in B. One of three identical experiments done in triplicate with similar results is shown. Control values forB and D represent the amount of tubulin associated with the plasma membrane in the absence of carbachol or PIP₂.