Structural determinants for phosphatidic acid regulation of phospholipase C-β1

Elliott M. Ross, Dania Mateu, Aldrin V Gomes, Carlos Arana, Thanh Tran, Irene Litosch

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

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 Gαq 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 Gαq binding domain. The results identify a region that includes the αA helix and flexible loop of the Gαq-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 Gαq-binding domain. PA regulation of PLC-β1 requires unique residues that are not required for Gαq stimulation or GTPase-activating protein activity.

Original languageEnglish (US)
Pages (from-to)33087-33094
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number44
DOIs
StatePublished - Nov 3 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Ross, E. M., Mateu, D., Gomes, A. V., Arana, C., Tran, T., & Litosch, I. (2006). Structural determinants for phosphatidic acid regulation of phospholipase C-β1. Journal of Biological Chemistry, 281(44), 33087-33094. https://doi.org/10.1074/jbc.M606487200