Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A

Abstract

Phospholipase Cε (PLCe) is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with small GTPases, including Rap1A and Ras. While Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability, or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors (β-ARs), translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by 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 also necessary for activation. Finally, small angle X-ray scattering (SAXS) showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. This data, together with the recent structure of a catalytically active fragment of PLCe, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLC core.

Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation

“…Our studies with TSHR are consistent with Ca 2+ as the main intracellular factor regulating nuclear sAC-dependent cAMP accumulation using a Gαq-independent PLC-InsP3-InsP3R pathway. Among the PLC isoforms, a likely candidate is PLCε, a GPCR-activated isoform that is modulated by Rap1-GTP, that binds directly to and allosterically modulates PLCε ( 87 ), but not Gαq ( 88 , 89 ). Furthermore, it was reported that PLCε resides in perinuclear membranes near the nuclear envelope ( 90 , 91 ) and has previously been shown to mediate Epac1-Rap1 stimulatory effects on InsP3 production and Ca 2+ release ( 92 – 95 ).…”