Abstract
Signaling from G protein-coupled receptors to phospholipase C-beta (PLC-beta) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-beta(1) through a mechanism that requires the PLC-beta(1) C-terminal domain. PA also modulates Galpha(q) stimulation of PLC-beta(1). These data suggest that PA may have a key role in the regulation of PLC-beta(1) signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-beta(1). We used a combination of enzymatic assays, PA-binding assays, and circular dichroism spectroscopy to evaluate the interaction of PA with wild-type and mutant PLC-beta(1) proteins and with fragments of the Galpha(q) binding domain. The results identify a region that includes the alphaA helix and flexible loop of the Galpha(q)-binding domain as necessary for PA regulation. A mutant PLC-beta(1) with multiple alanine/glycine replacements for residues (944)LIKEHTTKYNEIQN(957) was markedly impaired in PA regulation. The high affinity and low affinity component of PA stimulation was reduced 70% and PA binding was reduced 45% in this mutant. Relative PLC stimulation by PA increased with PLC-beta(1) concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-beta(1) mutant. These data are consistent with a model for PA regulation of PLC-beta(1) that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Galpha(q)-binding domain. PA regulation of PLC-beta(1) requires unique residues that are not required for Galpha(q) stimulation or GTPase-activating protein activity.
Highlights
EXPERIMENTAL PROCEDURESExpression and Purification of C-terminal Fragments— PLC-1 exists as two alternatively spliced variants, 150-kDa PLC-1a and 140-kDa PLC-1b, that are comparably stimulated by G␣q
Solving the crystal structure of the turkey PLC- C terminus required removal of 76% of the turkey flexible loop [30]. This corresponds to rat PLC-1 residues 961–993 and lies outside the sequence required for PA regulation as identified in the present study
The data presented here suggest that PLC-1 dimerization, specific PA binding, and stimulation of phospholipase activity are allosterically coupled
Summary
Expression and Purification of C-terminal Fragments— PLC-1 exists as two alternatively spliced variants, 150-kDa PLC-1a and 140-kDa PLC-1b, that are comparably stimulated by G␣q. PLC fragments or PLC-1 were added to a 100-l assay mixture (liposomes consisting of 400 M 100 mol % PC or 25 mol % PA/75 mol % PC or, as indicated, 30 mM HEPES (pH 7.0), 5 g of fatty acid-free bovine serum albumin plus 0.1 M KCl or, as indicated, final concentration). The lipid-bound protein was separated from free protein by centrifugation, the pellet was resuspended in 25 mM Tris (pH 7.0) diluted with 4ϫ SDSPAGE sample buffer, and proteins were resolved by electrophoresis on 10 –20% SDS-PAGE (for C-terminal fragments) or 7.5% SDS-PAGE (for PLC-(1– 880) and PLC-1). PLC-1 was incubated in 50 l of buffer (0.5 mM MgCl2, 100 mM KCl, 50 mM HEPES (pH 7.0)) and 150 M phospholipid vesicles consisting of 50 M [3H]PIP2 in combination with PC and PA to achieve the appropriate mol % PA. A value of p Ͻ 0.05 was considered significant
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