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bioRxiv · 10.1101/2020.07.10.191643

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

Monita Sieng·Arielle F. Selvia·Elisabeth E. Garland-Kuntz·Jesse B. Hopkins·Isaac J. Fisher·Andrea T. Marti·Angeline Lyon
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Abstract

Phospholipase C{varepsilon} (PLC{varepsilon}) 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 PLC{varepsilon} in response to the stimulation of {beta}-adrenergic receptors ({beta}-ARs), translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLC{varepsilon} 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 PLC{varepsilon} 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 by the GTPase. Finally, small angle X-ray scattering (SAXS) showed that Rap1A binding induces and stabilizes discrete conformational states in PLC{varepsilon} variants that can be activated by the GTPase. This data, together with the recent structure of a catalytically active fragment of PLC{varepsilon}, 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.

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Monita Sieng, Arielle F. Selvia, Elisabeth E. Garland-Kuntz, Jesse B. Hopkins, Isaac J. Fisher, Andrea T. Marti, Angeline Lyon. 2020-07-10. Functional and structural characterization of allosteric activation of Phospholipase Cϵ by Rap1A. https://doi.org/10.1101/2020.07.10.191643

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