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Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state.

ABSTRACT: Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On- and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low-affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high-affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by 'inside-out signaling.'

SUBMITTER: Li J

PROVIDER: S-EPMC8730728 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state.

Li Jing J Yan Jiabin J Springer Timothy A TA

eLife 20211202

Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that ...[more]

PMID: 34854380

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Project description:Backgroundα3β1 integrin is a promising cancer biomarker and drug target. We previously identified a 9-amino-acid cyclic peptide LXY30 for detecting α3β1 integrin on the surface of live tumor cells. This study was undertaken to characterize LXY30 in the detection, cellular function, imaging, and targeted delivery of in vitro and in vivo non-small cell lung cancer (NSCLC) models.MethodsThe whole-cell binding assay was performed by incubating NSCLC cells, extracellular vesicles (EVs), and peripheral blood mononuclear cells (PBMCs) with TentaGel resin beads coated with LXY30. In this study, we defined the nanosize EVs as exosomes, which were characterized by flow cytometry, transmission electron microscopy, dynamic light scattering, and Western blots. The function of LXY30 was determined by modulating the epidermal growth factor receptor (EGFR) signaling pathway by growth inhibition and Western blots. For in vivo biodistribution, mice bearing subcutaneous and intracranial NSCLC xenograft tumors were administrated intraveneously with LXY30-biotin/streptavidin-Cy5.5 complex and then analyzed for in vivo and ex vivo optical imaging and histopathology.ResultsWe showed that LXY30 specifically and sensitively detected α3β1 integrin-expressing NSCLC cells and tumor-derived exosomes. Tumor DNA isolated from LXY30-enriched plasma exosomes might be used to detect driver oncogenic mutations in patients with metastatic NSCLC. LXY30 only enriches tumor cells but not neutrophils, macrophages, or monocytes in the malignant pleural effusion of NSCLC patients for detecting genomic alterations by next-generation sequencing. LXY30 detected increased α3β1 integrin expression on the EGFR-mutant NSCLC cells with acquired resistance to erlotinib compared to parental erlotinib-sensitive EGFR-mutant NSCLC cells. We further showed that LXY30 modulated the EGFR signaling pathway independently from another peptide ligand LXW64 targeting αvβ3 integrin in erlotinib-resistant, EGFR-mutant H1975 cells. Analysis of The Cancer Genome Atlas (TCGA) revealed high α3 integrin expression was associated with poor prognosis in lung squamous cell carcinoma. LXY30-biotin/streptavidin-Cy5.5 complex had higher uptakes in the subcutaneous and intracranial xenografts of various α3β1 integrin-expressing lung adenocarcinoma and patient-derived lung squamous cell carcinoma xenografts while sparing the surrounding normal tissues.ConclusionLXY30 is a promising peptide for the cancer diagnosis and in vivo targeted delivery of imaging agents and cancer drugs in NSCLC, independent of histology and tumor genotype.

Dataset Information

Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state.

Publications

Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state.

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