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Structural determinants for phosphatidic acid regulation of phospholipase C-beta1.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2006 Nov 03; Vol. 281 (44), pp. 33087-94. Date of Electronic Publication: 2006 Sep 01. - Publication Year :
- 2006
-
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.
- Subjects :
- Binding Sites
Circular Dichroism
Enzyme Activation
Isoenzymes genetics
Mutation genetics
Phenotype
Phospholipase C beta
Type C Phospholipases genetics
Isoenzymes chemistry
Isoenzymes metabolism
Phosphatidic Acids chemistry
Phosphatidic Acids metabolism
Type C Phospholipases chemistry
Type C Phospholipases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 281
- Issue :
- 44
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 16950781
- Full Text :
- https://doi.org/10.1074/jbc.M606487200