Campus Units

Chemistry

Document Type

Article

Publication Version

Published Version

Publication Date

3-9-2007

Journal or Book Title

Analytical Chemistry

Volume

79

Issue

8

First Page

3142

Last Page

3147

DOI

10.1021/ac062008i

Abstract

A method for measuring the microclustering of a class of cell surface receptors called integrins is reported. Integrins are proteins involved in bidirectional signaling across the cell membrane and are important in cell adhesion, growth, and survival. Their activity is regulated by changes in protein conformation and protein clustering. The developed in vivo clustering assay uses fluorescence resonance energy transfer (FRET) and has the benefit of requiring a single cloning step to generate FRET donors and acceptors that can be used to measure the clustering of a series of integrin mutants. The FRET reporters contain extracellular donor or acceptor fluorescent protein attached to native integrin cytoplasmic and transmembrane domains, and these are expressed along with wild-type or mutant integrins. Expression of the FRET reporters has no affect on the ligand binding properties of coexpressed integrins. FRET values are calculated for cell lines spreading on ligand coated surfaces, and these values are independent of fluorescent protein expression. No FRET is observed in cell lines expressing the reporters in the absence of integrins. Integrin-dependent FRET values increase ∼2−3-fold when the integrins contain mutations that result in increased ligand binding affinities.

Comments

Reprinted (adapted) with permission from Analytical Chemistry, 79(8); 3142-3147. Doi: 10.1021/ac062008i. Copyright 2007 American Chemical Society.

Copyright Owner

American Chemical Society

Language

en

File Format

application/pdf

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