Coney, George
(2025)
Elucidation of Potential NRP1 Interactors using BioID Technology.
PhD thesis, University of Nottingham.
Abstract
Background:
Neuropilin 1 (NRP1) is a transmembrane glycoprotein involved in diverse physiological processes, including angiogenesis, axonal guidance, immune regulation, cell adhesion, and recently, facilitation of SARS-CoV-2 entry and infectivity. Despite its functional importance, comprehensive analyses of the NRP1 interactome have been limited. Previous studies focused primarily on the N-terminal region, while the intracellular C-terminal portion, containing a PDZ domain, remains largely unexplored. Identifying NRP1 binding partners, particularly those interacting with its C-terminal region, is critical to fully understand its cellular roles.
Aims:
This study aimed to define the NRP1 interactome using BioID proximity labeling technology, both under basal conditions and following stimulation with its cognate ligand VEGF165a. A particular focus was placed on characterizing interactions mediated by the intracellular C-terminal region. Selected candidate interactors were further validated using NanoLuciferase Bioluminescence Resonance Energy Transfer (NanoBRET) assays.
Results:
In HEK293T cells, BioID analysis identified 177 proteins enriched under basal conditions and 42 potential interactors upon VEGF165a stimulation. Among a subset of proteins selected for further validation—Vesicle-associated membrane protein-associated protein A (VAPA), Occludin (OCLN), Zona occludens 1 (ZO1), Septin 9 (SEPT9), Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), and Integrin β1 (ITGB1)—positive interactions were particularly observed for OCLN and VAPA, associated with cell adhesion and vesicular trafficking, respectively. Further experiments using hTERT2-HUVEC cells, a more physiologically relevant endothelial model, revealed additional potential interactors, suggesting that NRP1 may exhibit context-dependent, cell-specific interactions.
Conclusions:
This study presents the most detailed analysis to date of the NRP1 interactome, highlighting the role of its C-terminal region in recruiting PDZ domain-containing proteins. BioID combined with NanoBRET validation demonstrates that NRP1 interactions are both ligand-dependent and cell-type-specific, particularly involving proteins implicated in adhesion and trafficking. These findings provide a foundation for understanding the molecular mechanisms underlying NRP1 function in diverse physiological and pathological contexts.
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