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Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells

PNAS, 26 April 2011
Vol. 108, Issue 17, p. 7016-7021
DOI: 10.1073/pnas.1018280108

Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells

  1. Jody L. Swifta,1,
  2. Antoine G. Godinb,1,
  3. Kim Doréc,
  4. Laure Frelandc,
  5. Nathalie Bouchardc,
  6. Chelsea Nimmoa,
  7. Mikhail Sergeevb,
  8. Yves De Koninckc,
  9. Paul W. Wisemana,b, and
  10. Jean-Martin Beaulieuc,2
  1. Departments of aChemistry and
  2. bPhysics, McGill University, Montreal, QC, Canada H3G 0B1; and
  3. cDepartment of Psychiatry and Neuroscience, Université Laval and Centre de Recherche Université Laval Robert-Giffard (CRULRG), Québec, QC, Canada G1R 2G3
  1. Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 14, 2011 (received for review December 13, 2010)

Abstract

Cell signaling involves dynamic changes in protein oligomerization leading to the formation of different signaling complexes and modulation of activity. Spatial intensity distribution analysis (SpIDA) is an image analysis method that can directly measure oligomerization and trafficking of endogenous proteins in single cells. Here, we show the use of SpIDA to quantify dimerization/activation and surface transport of receptor protein kinases—EGF receptor and TrkB—at early stages of their transactivation by several G protein-coupled receptors (GPCRs). Transactivation occurred on the same timescale and was directly limited by GPCR activation but independent of G-protein coupling types. Early receptor protein kinase transactivation and internalization were not interdependent for all receptor pairs tested, revealing heterogeneity between groups of GPCRs. SpIDA also detected transactivation of TrkB by dopamine receptors in intact neurons. By allowing for time and space resolved quantification of protein populations with heterogeneous oligomeric states, SpIDA provides a unique approach to undertake single cell multivariate quantification of signaling processes involving changes in protein interactions, trafficking, and activity.

  • high content analysis
  • oligomers
  • monoamine
  • N-acetyl cystein
  • brain-derived neurotrophic factor

Footnotes

  • 1J.L.S. and A.G.G. contributed equally to this work.

  • 2To whom correspondence should be addressed. E-mail: martin.beaulieu{at}crulrg.ulaval.ca.
  • Author contributions: J.L.S., A.G.G., K.D., L.F., Y.D.K., P.W.W., and J.-M.B. designed research; J.L.S., A.G.G., K.D., L.F., N.B., C.N., M.S., and J.-M.B. performed research; P.W.W. and J.-M.B. contributed new reagents/analytic tools; J.L.S., A.G.G., K.D., L.F., N.B., C.N., M.S., and J.-M.B. analyzed data; and J.L.S., A.G.G., Y.D.K., P.W.W., and J.-M.B. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1018280108/-/DCSupplemental.

Freely available online through the PNAS open access option.

Citation:

J. L. Swift, A. G. Godin, K. Doré, L. Freland, N. Bouchard, C. Nimmo, M. Sergeev, Y. De Koninck, P. W. Wiseman, and J.-M. Beaulieu, Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells. PNAS 108, 7016-7021 (2011).

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