Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology. In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation. The Gip17 protein was purified to homogeneity from D. discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase. NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins. We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein. The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D. melanogaster development. The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity. Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential. Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation.

Cells of Dictyostelium discoideum respond chemotactically to.cyclic AMP [l] and to folic acid [3]. Cyclic AMP is a most efficient attractant for cells of the aggregation phase [2], folic acid is more effective with preaggregation cells [4]. Both cyclic AMP and folic acid stimulate also cell development from the preaggregation phase to the aggregation competent state [S-8]. During this process the cells acquire the capacity to synthesize cyclic AMP periodically, and to release it into the extracellular space in form of pulses [9] . The administration of cyclic AMP or folic acid pulses accelerates the onset of sustained oscillations [5,8] .The cyclic-AMP induced responses are known to be mediated by cell-surface receptors [lo-121. The intermediate steps in signal processing from the receptors to the various intracellular targets are unknown. In the present communication we show that upon stimulation of early preaggregation cells with folic acid a rapid increase of the cyclic GMP concentration is induced. A biphasic increase of cyclic GMP is observed in late preaggregation cells stimulated by cyclic AMP. The second cyclic GMP peak is succeeded by a cyclic AMP peak known to be based on the activation of adenylate cyclase [ 131. Free-running oscillations of cyclic GMP were observed together with the periodic formation of cyclic AMP pulses, and the cyclic GMP peaks seemed to occur slightly in advance of the cyclic AMP peaks.

We used a Ca'-sensitive electrode to measure changes in extracellular Ca" concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation . The cells maintained an external level of 3-8 AM Ca" until the beginning of aggregation and then started to take up Ca". The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca" by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2

No abstract

The chemoattractant folic acid binds reversibly to receptors at the surface of Dictyostelium cells. In undifferentiated cells (to.5) 6 x lo4 binding sites per cell with a Ko.S value of 1.5 x M were determined. The number of folic acid receptors per cell decreases slightly during cell development. In differentiated cells (tlo) 3.3 x lo4 binding sites per cell were estimated. The folic acid binding sites appear to be specific for folic acid and its derivatives. 2-Deamino-2-hydroxyfolic acid, 4-aminofolic acid (aminopterin), and 4-amino-10-methylfolic acid (aniethopterin) compete for the folic-acidbinding sites. Pterins, p-aniinobenzoic acid and glutamic acid show no significant competition for the folic-acid-binding sites. Nor do adenosine 3',5'-phosphate and guanosine 3',5'-phosphate affect binding of folic acid. The folic acid receptors appear to be distinct from the catalytic sites of the membrane-bound folic acid deaminase.Cells of Dictyostelium discoideum react chemotactically to cyclic AMP [l] as well as to folic acid and pterins [2,3] We investigated the binding of folic acid to cells of D. discoideum and provide evidence for the presence of folic acid receptors at the cell's surface. The number of folic acid receptors per cell decreases slightly during cell development. The receptors appear to be specific: folic acid derivatives compete for the folic-acid-binding sites, but pterins do not compete. Also cyclic nucleotides do not influence binding of folic acid. An account of this work was published in abstract form [ll].