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A proteomics approach to isolating neuropilin-dependent α5 integrin trafficking pathways: neuropilin 1 and 2 co-traffic α5 integrin through endosomal p120RasGAP to promote polarised fibronectin fibrillogenesis in endothelial cells

ABSTRACT: Integrin trafficking to and from membrane adhesions is a crucial mechanism that dictates many aspects of a cell’s behaviour, including motility, polarisation, and invasion. In endothelial cells (ECs), the intracellular traffic of α5-integrin is regulated by both neuropilin 1 (NRP1) and neuropilin 2 (NRP2), yet the redundancies in function between these co-receptors remain unclear. Moreover, the endocytic complexes that participate in NRP-directed-traffic remain poorly annotated. Here we identify an important role for the GTPase-activating protein p120RasGAP in ECs, promoting the recycling of α5-integrin from early endosomes. Mechanistically, p120RasGAP enables transit of endocytosed α5-integrin-NRP1-NRP2 complexes to Rab11+ recycling endosomes, promoting cell polarisation and fibronectin (FN) fibrillogenesis. Silencing of both NRP receptors, or p120RasGAP, resulted in the accumulation of α5-integrin in early endosomes, a loss of α5-integrin from surface adhesions, and attenuated EC polarisation. Endothelial-specific deletion of both NRP1 and NRP2 in the postnatal retina recapitulated our in vitro findings, severely impairing FN fibrillogenesis and polarised sprouting. Our data assign an essential role for p120RasGAP during integrin traffic in ECs and support a hypothesis that NRP receptors co-traffic internalised cargoes. Importantly, we utilise comparative proteomics analyses to, for the first time, isolate a comprehensive map of NRP1-dependent and NRP2-dependent-α5-integrin interactions in ECs.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Lung

SUBMITTER: Douglas Lamont

LAB HEAD: Stephen Robinson

PROVIDER: PXD051778 | Pride | 2024-05-20

REPOSITORIES: Pride

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			Action	DRS
	20231111_075024_297_2023_CB_mouse_directDIA_v17.sne	Other
	297_2023_CB_mouse_directDIA_v17_AnalyisOverview.txt	Txt
	297_2023_CB_mouse_directDIA_v17_ConditionSetup.tsv	Tabular
	297_2023_CB_mouse_directDIA_v17_ExperimentSetupOverview_directDIA_no_profiling_no_imputation_FP_v16.txt	Txt
	297_2023_CB_mouse_directDIA_v17_Report_Protein_pivot_FP.xls	Xls

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Project description:Important remodeling of the extracellular matrix is a hallmark of corneal wound healing. Besides the massive secretion of fibronectin (FN) that characterizes the early step of that process, alterations in the secretion of collagens also occurs as part of this wound healingresponse. In this study, we examined whether expression of the gene encoding the α5 subunit from the FN binding integrin α5β1 changes as corneal epithelial cells (CECs) are cultured in the presence of collagens. Responsiveness of the α5 gene toward collagen was determined by transfection of recombinant plasmids bearing the CAT reporter gene under the control of various segments from the human α5 promoter into primary cultured rabbit (RCECs) and human (HCECs) CECs cultured on BSA or on collagens. Electrophoretic mobility shift assays (EMSAs) and Western blot analyses were used to monitor both the DNA binding and expression of the transcription factors required to ensure basal transcription of the α5 gene. The influence collagens on the patterns of genes expressed by HCECs was also studied be gene profiling on microarrays. All collagen types repressed the transcriptional activity directed by the full-length α5 promoter/CAT construct in confluent CECs. A moderate increase in α5 promoter activity was observed in subconfluent RCECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in either the expression or the DNA binding of the transcription factors Sp1/Sp3, NFI and AP-1 that ensure basal transcription of the α5 gene. Gene profiling on microarray revealed that CI more profoundly alter the pattern of genes expressed by HCECs than what is observed with CIV. Collagens considerably suppressed expression of the α5 gene in CECs at confluence suggesting that the sustained staining for CI and CIV after corneal injury may play a role in controlling adhesion to the temporary FN matrix and further differentiation of CECs into suprabasal epithelial cells through a mechanism involving the α5β1 integrin.

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A proteomics approach to isolating neuropilin-dependent α5 integrin trafficking pathways: neuropilin 1 and 2 co-traffic α5 integrin through endosomal p120RasGAP to promote polarised fibronectin fibrillogenesis in endothelial cells

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