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Unraveling the role of membrane proteins Notch, Pvr, and EGFR in altering integrin diffusion and clustering

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Abstract

The role of three membrane proteins in altering the diffusion and clustering of integrin receptors has been measured. Integrins are membrane proteins responsible for integrating intracellular and extracellular signaling events and anchoring cells to the extracellular matrix. The methodology used to elucidate the role of other membrane proteins in altering integrin diffusion and clustering combines fluorescence microscopy with RNA interference (RNAi), which is a technique to reduce the expression of a target protein. The three RNAi-targeted membrane proteins were epidermal growth factor receptor (EGFR), platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr), and Notch. Real-time polymerase chain reaction or quantitative immunocytochemistry was used to measure a reduction in mRNA or protein concentration after RNAi treatment, respectively. Fluorescence recovery after photobleaching showed that reducing the concentration of EGFR or Notch results in less constrained integrin diffusion and, in the case of Notch RNAi, 4 % more mobile integrins. Fluorescence resonance energy transfer measurements performed before and after RNAi treatments indicate that clustering decreases for wild-type integrin, but increases for a high-ligand-affinity integrin mutant after reducing the expression of EGFR, Pvr, or Notch. A model to explain the measured changes after reducing the expression of these three membrane proteins involving cholesterol-enriched nanodomains is proposed.

A combination of fluorescence microscopy and RNAi interference were used to measure how selectively reducing the expression of one membrane protein affects clustering and diffusion of another membrane protein

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Acknowledgments

This work was supported by the National Science Foundation (CHE-0845236). The authors thank Roger Tsien (Howard Hughes Medical Institute, La Jolla, CA) for the original mCherry plasmid, Atsushi Miyawaki (Riken, Wako-city, Saitama, Japan) for the original Venus plasmid, and Aasim Azad and Andrew Pavel (Iowa State University) for technical assistance. The RT-PCR experiments were performed at the Genomic Technologies Facility in the Center for Plant Genomics at Iowa State University. The monoclonal antibody developed by S. Artavanis Tsakonas (C458-2H) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

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  1. Department of Chemistry, Iowa State University, 1605 Gilman Hall, Ames, IA, 50011, USA

    Neha Arora, Dipak Mainali & Emily A. Smith

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  1. Neha Arora
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  2. Dipak Mainali
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Correspondence to Emily A. Smith.

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Arora, N., Mainali, D. & Smith, E.A. Unraveling the role of membrane proteins Notch, Pvr, and EGFR in altering integrin diffusion and clustering. Anal Bioanal Chem 404, 2339–2348 (2012). https://doi.org/10.1007/s00216-012-6362-3

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  • DOI: https://doi.org/10.1007/s00216-012-6362-3

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