In D. discoideum, cyclic AMP acting extracellularly as a primitive hormone directs a highly organized chemotactic aggregation of 10 to the six cells to form a multicellular structure. The nucleotide is periodically released from aggregation centers and diffuses to nearby cells where it binds to high affinity surface receptors and activates adenylate cyclase, thereby extending the range of the signal. The resulting cell-to-cell relay of the chemoattractant is detected in situ as propagated cAMP waves. This cAMP signaling system provides a biochemically and genetically accessible model for cell-cell communication, biological oscillations, eucaryotic chemotaxis and transmembrane signaling. An established perfusion assay which monitors cAMP elicited cAMP secretion will be used to study: 1) a defined set of cAMP analogues substituted at positions previously shown to alter chemotactic activity; 2) folic acid and related pteridines, alternate chemoattractants which potentiate the cAMP signaling response 2- to 3-fold; 3) concanavalin A which rapidly blocks 3H-cAMP secretion but not concomitant cGMP production. Recently discovered conditions for solubilization of adenylate cyclase have made possible the investigation of its physical properties in wild-type and mutant cells. One approach to the mechanism of regulation of the enzyme focuses on GTP binding, cholera toxin substrates (MW=42,000 and 44,000) which may represent a G/F regulatory protein similar to that found in vertebrates. Another approach is designed to discover factors which influence the activated state of the enzyme observed when cells are sonicated following stimulation by cAMP. A screening protocol is presented which will select those aggregation minus mutants in which the defect can be traced to the cAMP signaling response. Linkage group and complementation analyses are planned to map the defects and estimate the number of genes essential to each phenotype.
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