[Show abstract][Hide abstract] ABSTRACT: The regulation of cell polarity plays an important role in chemotaxis. Previously, two proteins termed GbpC and GbpD were identified in Dictyostelium, which contain RasGEF and cyclic nucleotide binding domains. Here we show that gbpC-null cells display strongly reduced chemotaxis, because they are unable to polarise effectively in a chemotactic gradient. However, gbpD-null mutants exhibit the opposite phenotype: cells display improved chemotaxis and appear hyperpolar, because cells make very few lateral pseudopodia, whereas the leading edge is continuously remodelled. Overexpression of GbpD protein results in severely reduced chemotaxis. Cells extend many bifurcated and lateral pseudopodia, resulting in the absence of a leading edge. Furthermore, cells are flat and adhesive owing to an increased number of substrate-attached pseudopodia. This GbpD phenotype is not dependent on intracellular cGMP or cAMP, like its mammalian homolog PDZ-GEF. Previously we showed that GbpC is a high-affinity cGMP-binding protein that acts via myosin II. We conclude that cGMP activates GbpC, mediating the chemoattractant-induced establishment of cell polarity through myosin. GbpD induces the formation of substrate-attached pseudopodia, resulting in increased attachment and suppression of polarity.