Linked Life Data

12503627

Source:http://linkedlifedata.com/resource/pubmed/id/12503627

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1-4
pubmed:dateCreated
2002-12-30
pubmed:abstractText
We have developed a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent, stopped-flow mixing, we determined that 32D cells, murine hematopoietic precursor cells, can survive rapid mixing, even at the high flow rates necessary to achieve dwell times as short as 10 msec. In addition, 32D cells do not express any member of the ErbB family of receptors, providing a null background for studying this receptor family. We have established a series of stable, monoclonal 32D-derived cell lines that express the epidermal growth factor (EGF) receptor, ErbB2, or a combination of both at different ratios. Using these cell lines and a homogeneous fluorescent derivative of H22Y-mEGF modified with fluorescein at the amino terminus (F-EGF), we have measured association and dissociation of F-EGF with its receptor. Association was measured by following the time-dependent changes in fluorescence anisotropy after rapidly mixing cells at various cell densities with F-EGF at 1-15nM. Dissociation was measured both by chase experiments in which unlabeled EGF was mixed with cells pre-equilibrated with F-EGF or by dilution of cells equilibrated with F-EGF. Comparison of these dissociation experiments demonstrated that little or no ligand-induced dissociation occurs in the chase dissociation experiments. For each cell line, data from a series of association experiments and dilution dissociation experiments were subjected to global analysis using a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations, even in cells bearing only the EGF receptor. Increasing the relative expression of ErbB2 leads to an increase in the fraction of high affinity class receptors observed, without altering the total number of EGF binding sites.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1079-9893
pubmed:author
pubmed:issnType
Print
pubmed:volume
22
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
357-71
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed-meshheading:12503627-Anisotropy, pubmed-meshheading:12503627-Cell Line, pubmed-meshheading:12503627-Cell Membrane, pubmed-meshheading:12503627-Cell Survival, pubmed-meshheading:12503627-Epidermal Growth Factor, pubmed-meshheading:12503627-Fluorescence Polarization, pubmed-meshheading:12503627-Hematopoietic Stem Cells, pubmed-meshheading:12503627-Humans, pubmed-meshheading:12503627-Ligands, pubmed-meshheading:12503627-Protein Binding, pubmed-meshheading:12503627-Receptor, Epidermal Growth Factor, pubmed-meshheading:12503627-Receptor, erbB-2, pubmed-meshheading:12503627-Receptors, Cell Surface, pubmed-meshheading:12503627-Recombinant Proteins, pubmed-meshheading:12503627-Spectrometry, Fluorescence, pubmed-meshheading:12503627-Stress, Mechanical, pubmed-meshheading:12503627-Transfection
pubmed:articleTitle
A stopped-flow fluorescence anisotropy method for measuring hormone binding and dissociation kinetics with cell-surface receptors in living cells.
pubmed:affiliation
Vanderbilt University, Department of Biological Sciences, Nashville, TN 37235, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.