Abstract
Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.
Highlights
From the ‡Department of Physiology and Biophysics and the ¶Department of Anesthesiology, State University of New York at Stony Brook, Stony Brook, New York 11794-8661
The results of this study show that the Pleckstrin homology (PH) domain of phospholipase C-2 (PLC-2) is responsible for the low basal activity, the nonspecific membrane binding behavior, and the G␥-dependent activation of full-length native phospholipase C (PLC) 2
We propose a model in which activation of PLC results from contacts between the PH domain and the catalytic core
Summary
Materials—Rat PLC-2 was expressed in Sf9 cells and human recombinant PLC-␦1 in Escherichia coli as described previously [13, 14]. Pleckstrin Homology Domain of PLC-2 Confers G␥ Activation succinyl ester at a 1:1 probe:protein molar ratio, and membrane binding was followed by the large increase in coumarin fluorescence as freshly extruded large, unilamellar vesicles were added. Membrane binding was assessed by following the association of unlabeled protein to freshly prepared large, unilamellar vesicles doped with 0.1 mol % of 6-lauroyl-2-(dimethyamino)naphthalene (Laurodan, Molecular Probes, Inc.) In this case, binding was followed by the increase in emission intensity (ex ϭ 350 nm) and shift to higher emission energy scanning from 380 –560 nm (see Ref. 12). G␥ subunits were solubilized in neutral detergent (CHAPS) and labeled at a 1:1 probe:protein molar ratio with the fluorescence energy transfer donor 7-methoxycoumarin succinyl ester using the previously described procedures [16]. The PLC partner proteins were labeled with the nonfluorescent energy transfer acceptor, 4-(dimethylamino)phenylazophenyl-4-sulfonyl chloride succinyl ester (Molecular Probes, Inc.), which does not interfere with enzyme activity or the ability of PLC-2 and 2PH-L/␦1 to be activated by G␥ [16]
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