Biology subjects

Ellisdon, A. M.

Publications and source records attributed to Ellisdon, A. M..

4 recordsLinked to original sources

bioRxiv2026

Intracellular targeting of a light-activated GPCR elicits spatially selective signalling

G protein-coupled receptors (GPCRs) initiate unique cell responses dependent on their sub-cellular location. Major advances in our understanding of sub-cellular GPCR signalling have been driven by experimental approaches that manipulate receptor trafficking, block signalling at specific intracellular sites, or compare receptor signalling in response to ligands with differing membrane permeability. Here, we describe an optogenetic platform that allows a single stimulus to activate the same GPCR at different intracellular membranes, enabling direct comparisons of spatial signalling in intact cells. To achieve this, we have directed a light-activated GPCR chimera to endosomal, mitochondrial, Golgi and nuclear membranes using C-terminal targeting sequences. We demonstrate activation of cAMP and ERK signalling in response to light at all sites; however, each targeted receptor generates a unique transcriptional fingerprint dependent on its sub-cellular location. Our engineered optogenetic platform reveals that GPCRs targeted to distinct intracellular locations are self-sufficient signalling platforms and highlights GPCR positioning as a critical driver of signalling. TeaserControlling GPCR signals by light and location reveals how intracellular positioning shapes cellular responses.

pharmacology and toxicology↗

bioRxiv2024

Feed-forward stimulation of CAMK2 by the oncogenic pseudokinase PEAK1 generates a therapeutically "actionable" signalling axis in triple negative breast cancer

The PEAK family of pseudokinases, comprising PEAK1-3, are signalling scaffolds that play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screened for PEAK1 effectors by affinity purification and mass spectrometry, identifying calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promoted CAMK2D/G activation in TNBC cells via a novel feed-forward mechanism involving PEAK1/PLC{gamma}1/Ca2+ signalling and direct binding via a consensus CAMK2 interaction motif in the PEAK1 N-terminus. In turn, CAMK2 phosphorylated PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurposed RA306, a second generation CAMK2 inhibitor under pre-clinical development for treatment of cardiovascular disease. RA306 demonstrated on-target activity against CAMK2 in TNBC cells and inhibited PEAK1-enhanced migration and invasion in vitro. Moreover, RA306 significantly attenuated TNBC xenograft growth and blocked metastasis in a manner mirrored by CRISPR-mediated PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus, identify a novel mechanism for regulation of Ca2+ signalling and its integration with tyrosine kinase signals, and identify CAMK2 as a therapeutically actionable target downstream of PEAK1.

cancer biology↗

bioRxiv2021

The cryo-EM structure of the neurofibromin dimer reveals the molecular basis for von Recklinghausen disease

Neurofibromin (NF1) is a tumour suppressor mutated in neurofibromatosis type 1 (von Recklinghausen disease), one of the most common human genetic diseases(1). NF1 regulates cellular growth through suppressing the Rat Sarcoma (RAS) pathway and, accordingly, mutations in this protein drive numerous cancers, including melanoma, ovarian, breast and brain cancer(2, 3). Currently, however, the molecular basis for NF1 function remains to be understood. Here we address this problem and use cryogenic Electron Microscopy (cryo-EM) to determine the structure of fulllength NF1. The 640 kDa NF1 homodimer forms an extraordinary lemniscate ({infty}) shaped molecule that is ~30 nm in length and ~ 10 nm wide. Each NF1 monomer comprises an N-terminal HEAT-repeat domain (N-HEAT), a guanosine triphosphatase activating protein (GAP)-related domain (GRD), a Sec14 homologous and pleckstrin homologous module (SEC-PH), and a C-terminal HEAT domain (C-HEAT). The core NF1 scaffold is formed via a head-to-tail dimer of the N- and C-HEAT domains. This platform, which is responsible for interacting with more than 10 regulatory binding partners, comprises an extraordinary array of over 150 -helices. Analysis of these EM data revealed that the GRD and SEC-PH domain are highly mobile with respect to the core scaffold and could not initially be accurately placed in electron density. Strikingly, however, using 3D variability analysis we were able to identify a significant subpopulation of NF1 particles and determine the complete NF1 structure to 5.6 [A] resolution. These data revealed that the catalytic GRD and lipid binding SEC-PH domain are positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation in order to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Finally, it is suggested that approaches to combat NF1-linked diseases may include release of the autoinhibited state in order to improve NF1 catalytic efficiency.

biochemistry↗

bioRxiv2020

Structural analysis of the PTEN:P-Rex2 signalling node reveals how cancer-associated mutations coordinate to hyperactivate Rac1

The PTEN:P-Rex2 complex is one of the most commonly mutated signaling nodes in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cell proliferation. Here, using extensive crosslinking mass spectrometry and functional studies, we provide crucial mechanistic insights into PTEN:P-Rex2 complex assembly and co-inhibition. PTEN is anchored to P-Rex2 by interactions between the PTEN PDZ-BM tail and the second PDZ domain of P-Rex2. This interaction bridges PTEN across the P-Rex2 surface, occluding PTEN membrane-binding and PI(3,4,5)P3 hydrolysis. Conversely, PTEN both allosterically promotes an autoinhibited P-Rex2 conformation and occludes G{beta}{gamma} binding and GPCR activation. These insights allow us to define a new gain-of-function class of cancer mutations within the PTEN:P-Rex2 interface that uncouples PTEN inhibition of Rac1 signaling. These findings provide a mechanistic framework to understand the dysregulation of the PTEN:P-Rex2 signaling node in metastatic cancer.

biochemistry↗