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Sci. Signal., 3 May 2011
Vol. 4, Issue 171, p. ec122
[DOI: 10.1126/scisignal.4171ec122]

EDITORS' CHOICE

Methodology Separation of Activation and Internalization

Nancy R. Gough

Science Signaling, AAAS, Washington, DC 20005, USA

Stimulation and activation of cell surface receptors, such as receptor tyrosine kinases, involves receptor oligomerization and internalization. The ability to analyze individual living cells is key to understanding these dynamic properties. Swift et al. applied spatial intensity distribution analysis (SpIDA), which involves fitting fluorescence intensity histograms from confocal images, to assess the dimerization and internalization of the receptor tyrosine kinases, the epidermal growth factor receptor (EGFR), and the neurotrophin receptor TrkB, in response to transactivation by various G protein–coupled receptors. They validated their approach by comparing the results obtained by SpIDA to those obtained by fluorescence lifetime imaging microscopy (FLIM) in cells transfected with a green fluorescent protein (GFP)–tagged EGFR (and an mCherry-tagged EGFR for the FLIM analysis). The brightness of GFP-EGFR increased in response to EGF or in response to activation of five different GPCRs: angiotensin 1a receptor (ATa1R), β2 adrenergic receptor (β2AR), the D1 or D2 dopamine receptors (D1R, D2R), or the neurokinin 1 receptor (NK-1R). EGF stimulation produced the maximal amount of dimerization and, by increasing the amount of GPCR cotransfected into the cells, the amount of GPCR-induced dimerization could be increased, suggesting that the abundance of the GPCR may be a limiting factor in transactivation. Unexpectedly, D1R, D2R, and NK-1R triggered dimerization and internalization (loss of signal from the surface), which could be prevented by expression of a dominant-negative inhibitor of endocytosis, whereas ATa1R and β2AR induced EGFR dimerization without internalization. To verify that this technique could be applied to endogenous proteins, transactivation of endogenous TrkB by either D1R or D2R was monitored in striatal neuron cultures in which the TrkB was detected by immunofluorescence. Direct activation of TrkB by brain-derived neurotrophic factor (BDNF) and transactivation by dopamine receptor stimulation were detected as an increase in fluorescence indicative of TrkB dimerization. Not only will this technique be useful in studying the initial steps in cell surface receptor signaling, it has also revealed that transactivation of receptor tyrosine kinases does not always result in internalization, which has implications for selective signaling in response to different GPCR stimulation.

J. L. Swift, A. G. Godin, K. Doré, L. Freland, N. Bouchard, C. Nimmo, M. Sergeev, Y. De Koninck. P. W. Wiseman, J.-M. Beaulieu, Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells. Proc. Natl. Acad. Sci. U.S.A. 108, 7016–7021 (2011). [Abstract] [Full Text]

Citation: N. R. Gough, Separation of Activation and Internalization. Sci. Signal. 4, ec122 (2011).



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