All living cells can sense their environment. The term directional sensing refers to the ability of a cell to determine the direction and proximity of an extracellular stimulus. Directional sensing is needed to detect morphogens that control differentiation and attractants that direct cell migration, as in chemotaxis. This fascinating response is critical in embryogenesis, angiogenesis, neuronal patterning, wound healing, and immunity. Chemotaxis is strikingly exhibited during the life cycle of the social amoebae, Dictyostelium discoideum. During growth, these cells track down and phagocytose bacteria. When starved, they move towards secreted adenosine 3?-5? cyclic monophosphate (cAMP) signals, form aggregates, and differentiate into spore and stalk cells. The fundamental role of chemotaxis in this simple eukaryote provides a powerful system for its analysis at both genetic and biochemical levels. Both amoebae and mammalian leukocytes use G protein-linked signaling pathways to respond to chemoattractants. Binding of the attractants to receptors of the seven transmembrane helix class leads to the dissociation of the G proteins into alpha and beta/gamma-subunits. Chemotaxis is likely mediated through the beta/gamma-subunits. In both leukocytes and amoebae, chemoattractants elicit a variety of rapid responses including transient increases in Ca2+ influx, in the intracellular messengers IP3, cAMP and guanosine 3?-5? cyclic monophosphate (cGMP), and in the phosphorylation of myosins I and II. Chemoattractants also induce actin polymerization, most likely through the activation of the Rho family of small guanosine trisphosphatases. All these events rapidly subside in the presence of persistent stimulation. This rapid inhibition may allow a migrating cell to "subtract" the ambient concentration of attractant and more accurately sense the direction of a gradient. This laboratory is interested in studying how specific G protein-coupled signaling events translate into complex cellular responses such as cell migration. We have shown that the essential regulator of adenylyl cyclase called CRAC, a pleckstrin homology (PH) domain-containing protein, rapidly and transiently translocates to the plasma membrane upon chemoattractant addition. Using the GFP technology, we have shown that this association occurs selectively at the leading edge of chemotaxing cells. These results suggest that the activation of adenylyl cyclase is spatially and temporally restricted. The exact mechanism of action by which CRAC binds the plasma membrane in such a specific fashion and activates adenylyl cyclase is unknown. In D. discoideum and mammalian cells, PKB u another PH domain-containing protein - also transiently associates with the leading edge of chemotaxing cells. We propose that cells target these PH domain-containing proteins to the plasma membrane in order to spatially regulate signal transduction pathways. Our goal is to identify the components involved in the targeting of these proteins and define the molecular mechanisms cells use to spatially activate signaling pathways as observed in chemotaxis.