Biology subjects

Garland-Kuntz, E. E.

Publications and source records attributed to Garland-Kuntz, E. E..

3 recordsLinked to original sources

bioRxiv2026

Gβγ engages PLCβ3 at multiple sites to reorient and facilitate its activation

Phospholipase C {beta} (PLC{beta}) enzymes are activated by heterotrimeric G protein subunits, increasing hydrolysis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. All four human PLC{beta} isoforms (PLC{beta}1-4) are activated by Gq, while PLC{beta}1-3 are activated to varying extents by G{beta}{gamma}. The binding sites for Gq on PLC{beta} are well-established and much has been learned about its mechanism of activation, but comparatively little is known about G{beta}{gamma}-dependent activation. In this work, we used cryo-electron microscopy (cryo-EM) single particle analysis (SPA), functional assays, and bioluminescence resonance energy transfer (BRET) to investigate how G{beta}{gamma} interacts with PLC{beta}3 in concert with activated Gq to regulate phospholipase activity. G{beta}{gamma} heterodimers bind multiple surfaces of PLC{beta}3 to promote activation but alone do not recruit the enzyme to the plasma membrane. Instead, G{beta}{gamma} facilitates activation by Gq, most likely by reorienting the phospholipase catalytic site at the membrane to maximize PI(4,5)P2 hydrolysis and downstream Ca2+ release. Cell-based functional assays demonstrate that G{beta}{gamma} is required for maximal PLC{beta}3 activation even when Gq heterotrimers are the sole source of G{beta}{gamma}. Together, these findings demonstrate that G{beta}{gamma} acts as a critical positive allosteric modulator that regularly acts in concert with Gq to activate PLC{beta}3 at the plasma membrane.

biochemistry↗

bioRxiv2024

RhoA Allosterically Activates Phospholipase Cε via its EF Hands

Phospholipase C{varepsilon} (PLC{varepsilon}) cleaves phosphatidylinositol lipids to increase intracellular Ca2+ and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLC{varepsilon} is activated via direct binding of small GTPases at the cytoplasmic leaflets of cellular membranes. In the cardiovascular system, the RhoA GTPase regulates PLC{varepsilon} to initiate a pathway that protects against ischemia/reperfusion injuries, but the underlying molecular mechanism is not known. We present here the cryo-electron microscopy (cryo-EM) reconstruction of RhoA bound to PLC{varepsilon}, showing that the G protein binds a unique insertion within the PLC{varepsilon} EF hands. Deletion of or mutations to this PLC{varepsilon} insertion decrease RhoA-dependent activation without impacting regulation by other G proteins. Together, our data support a model wherein RhoA binding to PLC{varepsilon} allosterically activates the lipase and increases its interactions with the membrane, resulting in maximum activity and cardiomyocyte survival.

biochemistry↗

bioRxiv2024

Cryo-EM Structure of Phospholipase Cϵ Defines N-terminal Domains and their Roles in Activity

Phospholipase C{varepsilon} (PLC{varepsilon}) increases intracellular Ca2+ and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLC{varepsilon} to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has also limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 [A] reconstruction of the largest fragment of PLC{varepsilon} to date in complex with an antigen binding fragment (Fab). The structure reveals that PLC{varepsilon} contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain form a continuous surface that could engage cytoplasmic leaflets of the plasma and perinuclear membranes, contributing to activity. Functional characterization of this surface confirm it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLC{varepsilon} and the first mechanistic insights into how it engages cellular membranes for activity.

biochemistry↗