# Mechanisms Underlying Regulation of PLCbeta by heterotrimeric G proteins

> **NIH NIH R01** · PURDUE UNIVERSITY · 2024 · $347,615

## Abstract

Phospholipase C b (PLCb) enzymes increase intracellular calcium in response to diverse extracellular signals,
regulating numerous processes including cell proliferation and survival. Dysregulation of their expression or
activity contributes to pathophysiological conditions such as heart disease, cancer, and addiction. All four PLCb
isoforms bind to the cytoplasmic leaflet of the plasma membrane where they hydrolyze phosphatidylinositol-4,5-
bisphosphate (PIP2). They are activated via direct interactions with the heterotrimeric G proteins Gaq, and in
most cases, by Gbg. All PLCb isozymes have two elements that profoundly autoinhibit PIP2 hydrolysis: a lid that
blocks access to the active site, and a helix in the proximal C-terminal domain that binds to the catalytic core. G
protein binding and recruitment of the lipase to a membrane surface has been proposed to displace all the
autoinhibitory elements, resulting in activation. However, recent cryo-electron microscopy (cryo-EM) structures
of G protein–PLCb3 complexes bound to model membranes reveal that this is model is insufficient. In these
structures, the catalytic core fails to engage the membrane and the active site remains blocked. Thus, the
mechanism of activation and the molecular basis of isoform-specific responses to G proteins remain unresolved.
Furthermore, G proteins may also increase PLCb activity by altering their behavior on the membrane surface,
an aspect that cannot be assessed via structures or cell-based assays alone. To address these gaps, we propose
a synergistic and interdisciplinary combination of functional and structural studies, cell-based assays, and single
molecule microscopy to investigate the structure and regulation of the four PLCb isoforms. In Aim 1, we use
functional analyses to test hypotheses derived from new structures of PLCb complexes. We will also determine
cryo-EM reconstructions of the four PLCb isoforms in solution, and a subset in complex with G proteins on model
membranes known to promote activity. Aim 2 utilizes bioluminescence resonance energy transfer (BRET) and
signaling assays to monitor the location and disposition of PLCb isoforms within a cell and measure the kinetics
of G protein-dependent activation in response to receptor stimulation. In Aim 3, single molecule total internal
reflection fluorescence (TIRF) microscopy will be used to dissect the contributions of the PLCb regulatory
domains, PIP2, and G proteins to the kinetic behavior of individual lipase molecules on model membranes. Strong
preliminary data is included in support of each aim. Our work will not only reveal new mechanistic insights in
PLCb regulation but allow us to identify regulatory features unique to each isozyme that could be targeted by
novel selective chemical probes.

## Key facts

- **NIH application ID:** 10988727
- **Project number:** 1R01GM152701-01A1
- **Recipient organization:** PURDUE UNIVERSITY
- **Principal Investigator:** Angeline Marie Lyon
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $347,615
- **Award type:** 1
- **Project period:** 2024-08-15 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10988727

## Citation

> US National Institutes of Health, RePORTER application 10988727, Mechanisms Underlying Regulation of PLCbeta by heterotrimeric G proteins (1R01GM152701-01A1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10988727. Licensed CC0.

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