Structural determinants for phosphatidic acid regulation of phospholipase C-β₁

Signaling from G protein-coupled receptors to phospholipase C-β (PLC-β) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-β₁ through a mechanism that requires the PLC-β₁ C-terminal domain. PA also modulates Gα_q stimulation of PLC-β₁. These data suggest that PA may have a key role in the regulation of PLC-β₁ signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-β₁. 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-β₁ proteins and with fragments of the Gα_q binding domain. The results identify a region that includes the αA helix and flexible loop of the Gα_q-binding domain as necessary for PA regulation. A mutant PLC-β₁ with multiple alanine/glycine replacements for residues ⁹⁴⁴LIKEHTTKYNEIQN⁹⁵⁷ 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-α₁ concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-β₁ mutant. These data are consistent with a model for PA regulation of PLC- β₁ that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Gα_q-binding domain. PA regulation of PLC-β₁ requires unique residues that are not required for Gα_q stimulation or GTPase-activating protein activity.