The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.When Dictyostelium discoideum amoebae are deprived of nutrients, they initiate a developmental program that results in the formation of a multicellular organism composed of differentiated cell types. The aggregation process that takes place early in development is driven by chemotaxis toward cyclic AMP (cAMP) (6), and one of the earliest events in development is the elaboration of the biochemical apparatus required for chemotaxis toward cAMP (for reviews see references 7, 18, and 23). Soon after starvation, the cells synthesize an adenylate cyclase, a cell surface cAMP receptor, an extracellular phosphodiesterase, and a specific phosphodiesterase inhibitor. These proteins, among others, function coordinately to allow the cells to aggregate by chemotaxis. Cells secrete pulses of cAMP every 5 to 6 min, and neighboring cells respond by moving toward elevated concentrations of cAMP and by emitting cAMP to create a relay. The binding of cAMP to cell surface receptors activates second messenger cascades involving G proteins (27) that lead to a variety of cellular events (for reviews, see references 13 and 20), including the chemotactic response and the regulation of genes specific for development. The cAMP receptor is down regulated and phosphorylated upon constant stimulation by cAMP (25,53), and this results in the adaptation of cellular responses (52). The cyclic nucleotide phosphodiesterase (phosphodiesterase) acts outside the cell to reduce cAMP levels, limiting saturation and down regulation of the receptor. The phosphodiesterase exists in membrane-bound and free extracellular forms (30, 47). Its activity is regulated at the gene level (16) and is controlled at the protein level by a specific inhibitor (15). A m...

No abstract

The development and cellular differentiation of Dictyostelium discoideum are disrupted in transformants secreting high levels of the cyclic nucleotide phosphodiesterase.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) gene plays essential roles in the development of Dictyostelium discoideum during cellular aggregation and postaggregation morphogenesis. Genomic clones spanning the gene were isolated and used to determine the sequence and structure of the phosphodiesterase gene. We found an unusually complex organization for a gene of D. discoideum. Two transcripts of 2.4 and 1.9 kilobases (kb) were synthesized from start sites separated by 1.1 kb. A developmentally regulated promoter was utilized for the 2.4-kb mRNA, and a constitutive promoter regulated synthesis of the 1.9-kb transcript. The gene was found to be divided into four exons that are alternately spliced to give rise to the two mRNAs. The precursor of the 2.4-kb mRNA contained a 2.3-kb intron, whereas the precursor of the constitutive transcript was synthesized with a 1.7-kb intron. The two transcripts contained identical protein-coding regions and 400-nucleotide 3' untranslated sequences. The 2.4-kb developmentally regulated mRNA was distinguished by a long 5' untranslated leader of 666 nucleotides. The complex structure of the gene may allow multiple levels of control of the expression of the phosphodiesterase during development.

The secreted cyclic nucleotide phosphodiesterase (PDE) and its glycoprotein inhibitor (PDI) are among the first genes expressed when Dictyostelium amoebae begin their development. We used a series of mutants with defects in signal transduction to ask whether cAMP receptors 1 and 3, G alpha2, G beta, adenylyl cyclase (ACA), or the protein kinase A catalytic subunit (PKAcat) are required for the initial appearance or later regulation of the PDE and the PDI transcripts. The PDE gene produces a 1.9-kb transcript during vegetative growth and then a 2.4-kb transcript during the early hours of development. Regulation of the 2.4-kb transcript in CAR1, G alpha2, G beta, and ACA mutants is similar to that of isogenic parental strains, although its level is reduced in strains that carry the CAR1 mutation. CAR1/CAR3 double mutants also produce less PDE transcript, but the PDE gene remains inducible by cAMP. The PKAcat mutant produces the 2.4-kb PDE transcript, but in this mutant the vegetative transcript is retained in development. CAR1 and CAR3 are not required for transcription of the PDI gene, but deleting CAR1 leads to overproduction of the PDI transcript. Induction or repression of the PDI gene does not require G alpha2, G beta, or ACA. PKAcat is required for synthesis of the PDI transcript. The results suggest a two-stage regulation of these early genes through a G alpha2/G beta-independent mechanism and an absolute dependence of PDI on the PKAcat.