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Vol. 16, Issue 2, 676-688, February 2005
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* Laboratories of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852;
Laboratories of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852
Submitted July 1, 2004;
Revised November 9, 2004;
Accepted November 11, 2004
Monitoring Editor: Paul Matsudaira
Activation of G-protein-coupled chemoattractant receptors triggers dissociation of G
and G
subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PHCrac-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When naïve cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PHCrac-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PHCrac-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Corresponding author. E-mail address: tjin{at}niaid.nih.gov.
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