ABSTRACT
Phospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with small GTPases. Following stimulation of β-adrenergic receptors (β-ARs), the Rap1A GTPase binds PLCε, translocating the complex to the perinuclear membrane. Although Ras has been reported to allosterically activate the enzyme, it is not known if Rap1A has the same ability or what its molecular mechanism might be. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed with purified proteins that the RA2 domain is indeed essential for high affinity interactions with Rap1A. However we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation, and identified hydrophobic residues on the surface of the RA2 domain that are necessary for activation but dispensable for GTPase binding. Finally, small angle X-ray scattering (SAXS) showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that are capable of being activated. Considered along with the recent structure of a catalytically active fragment of PLCε, our findings suggest that Rap1A and likely Ras allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLC core.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- PLC
- Phospholipase C
- RA
- Ras association
- DAG
- diacylglycerol
- IP3
- inositol-1,4,5-triphopshate
- PKC
- protein kinase C
- CHAPS
- _3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate
- GEF
- guanine exchange factor
- GPCR
- G protein-coupled receptor
- RTK
- receptor tyrosine kinase
- cAMP
- cyclic AMP
- Epac
- exchange protein activated by cAMP
- PIP
- phosphatidylinositol phosphate
- PIP2
- phosphatidylinositol (4,5)-bisphosphate
- PE
- phosphatidylethanolamine
- PH
- pleckstrin homology
- TIM
- triose-phosphate isomerase
- RA
- Ras association
- PI4P
- phosphatidylinositol-4-phosphate
- DSF
- differential scanning fluorimetry
- SAXS
- small angle X-ray scattering
- SEC
- size exclusion chromatography