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Vol. 16, Issue 2, 976-983, February 2005

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Activation of Soluble Guanylyl Cyclase at the Leading Edge during Dictyostelium Chemotaxis

Douwe M. Veltman ^*, Jeroen Roelofs ^* , Ruchira Engel , Antonie J.W.G. Visser , and Peter J.M. Van Haastert ^* 

^* Department of Biochemistry, University of Groningen, 9747 AG Groningen, The Netherlands; MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands

Submitted August 13, 2004; Revised November 26, 2004; Accepted November 29, 2004
Monitoring Editor: Peter Devreotes

Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn²⁺/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg²⁺/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg²⁺/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-5730.

Corresponding author. E-mail address: P.J.M.van.Haastert{at}rug.nl.

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